Anti-prlr antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to prolactin receptor (PRLR) and methods of using the same. According to certain embodiments, the antibodies of the invention bind human PRLR with high affinity. In certain embodiments, the invention includes antibodies that bind PRLR and block prolactin-mediated cell signaling. In other embodiments, the invention includes antibodies that bind PRLR but do not block prolactin-mediated cell signaling. The antibodies of the invention may be fully human antibodies. The invention includes anti-PRLR antibodies conjugated to a cytotoxic agent, radionuclide, or other moiety detrimental to cell growth or proliferation. The antibodies of the invention are useful for the treatment of various cancers as well as other PRLR-related disorders. The present invention also includes antibody drug conjugates comprising an antibody or antigen-binding fragment thereof that specifically binds a class I cytokine receptor, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 61/868,185, filed on Aug. 21, 2013;62/012,440, filed on Jun. 16, 2014; and 62/026,088, filed on Jul. 18,2014, the disclosures of which are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which specifically bind prolactin receptor (PRLR), aswell as antibody-drug conjugates comprising such antibodies, and methodsof use thereof.

BACKGROUND

Prolactin is a polypeptide growth hormone that exerts its activity byinteracting with the prolactin receptor (PRLR). PRLR is a singletransmembrane receptor belonging to the class 1 cytokine receptorsuperfamily. The binding of prolactin to PRLR leads to receptordimerization and intracellular signaling. Signaling through PRLR isassociated with various processes such as mammary gland development,lactation, reproduction and immunomodulation. Moreover, high levels ofPRLR expression have been detected in breast, prostate and other tumortypes.

Blockade of PRLR signaling has been suggested as a means for treatingbreast and prostate cancer. (See, e.g., Damiano and Wasserman, April2013, Clin. Cancer Res. 19(7):1644-1650). Anti-PRLR antibodies arementioned, e.g., in U.S. Pat. Nos. 7,867,493 and 7,422,899. Nonetheless,there is a need in the art for novel PRLR antagonists, such as anti-PRLRantibodies, for the treatment of cancer and other disorders associatedwith PRLR expression and/or signaling.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragmentsthereof that bind human prolactin receptor (PRLR). The antibodies of theinvention are useful, inter alia, for targeting tumor cells that expressPRLR. The anti-PRLR antibodies of the invention, and antigen-bindingportions thereof, may be used alone in unmodified form, or may beincluded as part of an antibody-drug conjugate or a bispecific antibody.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

Exemplary anti-PRLR antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs), light chainvariable regions (LCVRs), heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3), and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti-PRLRantibodies. Table 2 sets forth the nucleic acid sequence identifiers ofthe HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of theexemplary anti-PRLR antibodies.

The present invention provides antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising an HCVR comprising anamino acid sequence selected from any of the HCVR amino acid sequenceslisted in Table 1, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising an LCVRcomprising an amino acid sequence selected from any of the LCVR aminoacid sequences listed in Table 1, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity thereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising an HCVR and anLCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVRamino acid sequences listed in Table 1 paired with any of the LCVR aminoacid sequences listed in Table 1. According to certain embodiments, thepresent invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR/LCVR amino acid sequence pair containedwithin any of the exemplary anti-PRLR antibodies listed in Table 1. Incertain embodiments, the HCVR/LCVR amino acid sequence pair is selectedfrom the group consisting of: 18/26; 66/74; 274/282; 290/298; and370/378.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a heavy chainCDR1 (HCDR1) comprising an amino acid sequence selected from any of theHCDR1 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a heavy chainCDR2 (HCDR2) comprising an amino acid sequence selected from any of theHCDR2 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a heavy chainCDR3 (HCDR3) comprising an amino acid sequence selected from any of theHCDR3 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a light chainCDR1 (LCDR1) comprising an amino acid sequence selected from any of theLCDR1 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a light chainCDR2 (LCDR2) comprising an amino acid sequence selected from any of theLCDR2 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a light chainCDR3 (LCDR3) comprising an amino acid sequence selected from any of theLCDR3 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising an HCDR3 andan LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of theHCDR3 amino acid sequences listed in Table 1 paired with any of theLCDR3 amino acid sequences listed in Table 1. According to certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acidsequence pair contained within any of the exemplary anti-PRLR antibodieslisted in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acidsequence pair is selected from the group consisting of: 24/32; 72/80;280/288; 296/304; and 376/384.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind PRLR, comprising a set of sixCDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any ofthe exemplary anti-PRLR antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set is selected from the group consisting of:20-22-24-28-30-32; 68-70-72-76-78-80; 276-278-280-284-286-288;292-294-296-300-302-304; and 372-374-376-380-382-384.

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof that specifically bind PRLR,comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3)contained within an HCVR/LCVR amino acid sequence pair as defined by anyof the exemplary anti-PRLR antibodies listed in Table 1. For example,the present invention includes antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set containedwithin an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: 18/26; 66/74; 274/282; 290/298; and 370/378. Methods andtechniques for identifying CDRs within HCVR and LCVR amino acidsequences are well known in the art and can be used to identify CDRswithin the specified HCVR and/or LCVR amino acid sequences disclosedherein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-PRLR antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-PRLR antibodies listed inTable 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-PRLR antibodies listed inTable 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-PRLR antibody listed in Table1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-PRLR antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-PRLR antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds PRLR and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an anti-PRLR antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-PRLR antibody. The presentinvention also provides antibody-drug conjugates (ADCs) comprising ananti-PRLR antibody conjugated to a cytotoxic agent. Exemplarycombination therapies, co-formulations, and ADCs involving the anti-PRLRantibodies of the present invention are disclosed elsewhere herein.

In yet another aspect, the invention provides therapeutic methods forkilling tumor cells or for inhibiting or attenuating tumor cell growthusing an anti-PRLR antibody or antigen-binding portion of an antibody ofthe invention. The therapeutic methods according to this aspect of theinvention comprise administering a therapeutically effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment of an antibody of the invention to a subject in need thereof.The disorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by targeting PRLR and/or byinhibiting prolactin-mediated cell signaling through PRLR.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expression prolactin receptor, “PRLR,” and the like, as used herein,refers to the human prolactin receptor, comprising the amino acidsequence as set forth in SEQ ID NO:404. The expression “PRLR” includesboth monomeric and multimeric PRLR molecules. As used herein, theexpression “monomeric human PRLR” means a PRLR protein or portionthereof that does not contain or possess any multimerizing domains andthat exists under normal conditions as a single PRLR molecule without adirect physical connection to another PRLR molecule. An exemplarymonomeric PRLR molecule is the molecule referred to herein as“hPRLR.mmh” comprising the amino acid sequence of SEQ ID NO:401 (see,e.g., Example 3, herein). As used herein, the expression “dimeric humanPRLR” means a construct comprising two PRLR molecules connected to oneanother through a linker, covalent bond, non-covalent bond, or through amultimerizing domain such as an antibody Fc domain. An exemplary dimericPRLR molecule is the molecule referred to herein as “hPRLR.mFc”comprising the amino acid sequence of SEQ ID NO:402 (see, e.g., Example3, herein).

All references to proteins, polypeptides and protein fragments hereinare intended to refer to the human version of the respective protein,polypeptide or protein fragment unless explicitly specified as beingfrom a non-human species. Thus, the expression “PRLR” means human PRLRunless specified as being from a non-human species, e.g., “mouse PRLR,”“monkey PRLR,” etc.

As used herein, the expression “cell surface-expressed PRLR” means oneor more PRLR protein(s), or the extracellular domain thereof, thatis/are expressed on the surface of a cell in vitro or in vivo, such thatat least a portion of a PRLR protein is exposed to the extracellularside of the cell membrane and is accessible to an antigen-bindingportion of an antibody. A “cell surface-expressed PRLR” can comprise orconsist of a PRLR protein expressed on the surface of a cell whichnormally expresses PRLR protein. Alternatively, “cell surface-expressedPRLR” can comprise or consist of PRLR protein expressed on the surfaceof a cell that normally does not express human PRLR on its surface buthas been artificially engineered to express PRLR on its surface.

As used herein, the expression “anti-PRLR antibody” includes bothmonovalent antibodies with a single specificity, as well as bispecificantibodies comprising a first arm that binds PRLR and a second arm thatbinds a second (target) antigen, wherein the anti-PRLR arm comprises anyof the HCVR/LCVR or CDR sequences as set forth in Table 1 herein. Theexpression “anti-PRLR antibody” also includes antibody-drug conjugates(ADCs) comprising an anti-PRLR antibody or antigen-binding portionthereof conjugated to a drug or toxin (i.e., cytotoxic agent). Theexpression “anti-PRLR antibody” also includes antibody-radionuclideconjugates (ARCs) comprising an anti-PRLR antibody or antigen-bindingportion thereof conjugated to a radionuclide.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., PRLR). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-PRLR antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2 (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; V_(H)-C_(H)2-C_(H)3; (Vii) V_(H)-C_(L);(Viii) V_(L)-C_(H)1; (iX) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-PRLR antibodies of theinvention are human antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In 010-8198-3625/1/AMERICAS one form, an immunoglobulinmolecule comprises a stable four chain construct of approximately150-160 kDa in which the dimers are held together by an interchain heavychain disulfide bond. In a second form, the dimers are not linked viainter-chain disulfide bonds and a molecule of about 75-80 kDa is formedcomposed of a covalently coupled light and heavy chain (half-antibody).These forms have been extremely difficult to separate, even afteraffinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The anti-PRLR antibodies disclosed herein may comprise one or more aminoacid substitutions, insertions and/or deletions in the framework and/orCDR regions of the heavy and light chain variable domains as compared tothe corresponding germline sequences from which the antibodies werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-PRLR antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-PRLR antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences set forth in Table 1 herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

pH-Dependent Binding

The present invention includes anti-PRLR antibodies with pH-dependentbinding characteristics. For example, an anti-PRLR antibody of thepresent invention may exhibit reduced binding to PRLR at acidic pH ascompared to neutral pH. Alternatively, anti-PRLR antibodies of theinvention may exhibit enhanced binding to PRLR at acidic pH as comparedto neutral pH. The expression “acidic pH” includes pH values less thanabout 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6,5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, orless. As used herein, the expression “neutral pH” means a pH of about7.0 to about 7.4. The expression “neutral pH” includes pH values ofabout 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding to PRLR at acidic pH as comparedto neutral pH” is expressed in terms of a ratio of the K_(D) value ofthe antibody binding to PRLR at acidic pH to the K_(D) value of theantibody binding to PRLR at neutral pH (or vice versa). For example, anantibody or antigen-binding fragment thereof may be regarded asexhibiting “reduced binding to PRLR at acidic pH as compared to neutralpH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Anti-PRLR Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-PRLRantibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-PRLR antibodies comprising a mutation inthe C_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at position 250 and/or 428; or a modification at position307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P).

For example, the present invention includes anti-PRLR antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433Kand N434F). All possible combinations of the foregoing Fc domainmutations, and other mutations within the antibody variable domainsdisclosed herein, are contemplated within the scope of the presentinvention.

Biological Characteristics of the Antibodies

The present invention includes antibodies and antigen-binding fragmentsthereof that bind monomeric human PRLR with high affinity. For example,the present invention includes anti-PRLR antibodies that bind monomerichuman PRLR (e.g., hPRLR.mmh) with a K_(D) of less than about 4.0 nM asmeasured by surface plasmon resonance at 25° C. or 37° C., e.g., usingan assay format as defined in Example 3 herein, or a substantiallysimilar assay. According to certain embodiments, anti-PRLR antibodiesare provided that bind monomeric human PRLR at 37° C. with a K_(D) ofless than about 4 nM, less than about 3 nM, less than about 2 nM, lessthan about 1 nM, less than about 900 pM, less than about 800 pM, lessthan about 700 pM, less than about 600 pM, less than about 500 pM, lessthan about 400 pM, or less than about 300 pM, as measured by surfaceplasmon resonance, e.g., using an assay format as defined in Example 3herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind monomeric human PRLR (e.g., hPRLR.mmh) witha dissociative half-life (t %) of greater than about 5 minutes asmeasured by surface plasmon resonance at 25° C. or 37° C., e.g., usingan assay format as defined in Example 3 herein, or a substantiallysimilar assay. According to certain embodiments, anti-PRLR antibodiesare provided that bind monomeric human PRLR at 37° C. with a t½ ofgreater than about 5 minutes, greater than about 6 minutes, greater thanabout 8 minutes, greater than about 10 minutes, greater than about 12minutes, greater than about 14 minutes, greater than about 16 minutes,greater than about 18 minutes, greater than about 20 minutes, greaterthan about 30 minutes, greater than about 40 minutes, or longer, asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind dimeric human PRLR (e.g., hPRLR.mFc) withhigh affinity. For example, the present invention includes anti-PRLRantibodies that bind dimeric human PRLR with a K_(D) of less than about250 pM as measured by surface plasmon resonance at 25° C. or 37° C.,e.g., using an assay format as defined in Example 3 herein, or asubstantially similar assay. According to certain embodiments, anti-PRLRantibodies are provided that bind dimeric human PRLR at 37° C. with aK_(D) of less than about 250 pM, less than about 200 pM, less than about180 pM, less than about 160 pM, less than about 140 pM, less than about120 pM, less than about 100 pM, less than about 80 pM, less than about70 pM, or less than about 60 pM, as measured by surface plasmonresonance, e.g., using an assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind dimeric human PRLR (e.g., hPRLR.mFc) with adissociative half-life (t %) of greater than about 55 minutes asmeasured by surface plasmon resonance at 25° C. or 37° C., e.g., usingan assay format as defined in Example 3 herein, or a substantiallysimilar assay. According to certain embodiments, anti-PRLR antibodiesare provided that bind dimeric human PRLR at 37° C. with a t % ofgreater than about 55 minutes, greater than about 60 minutes, greaterthan about 65 minutes, greater than about 70 minutes, greater than about75 minutes, greater than about 80 minutes, greater than about 85minutes, greater than about 90 minutes, greater than about 95 minutes,greater than about 100 minutes, greater than about 120 minutes, greaterthan about 140 minutes, greater than about 160 minutes, or longer, asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind PRLR and block prolactin-mediated signalingin cells expressing human PRLR. For example, the present inventionincludes anti-PRLR antibodies that block prolactin-mediated signaling incells that express human PRLR, with an IC₅₀ of less than about 1.3 nM asmeasured using a prolactin signaling blocking assay, e.g., using anassay format as defined in Example 5 herein, or a substantially similarassay. According to certain embodiments, anti-PRLR antibodies areprovided that block prolactin-mediated signaling in cells expressinghuman PRLR, with an IC₅₀ of less than about 1.3 nM, less than about 1.2nM, less than about 1.0 nM, less than about 900 pM, less than about 800pM, less than about 600 pM, less than about 400 pM, less than about 200pM, less than about 100 pM, less than about 80 pM, less than about 60pM, less than about 40 pM, less than about 20 pM as measured using aprolactin signaling blocking assay, e.g., using an assay format asdefined in Example 5 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind PRLR but do not block prolactin-mediatedsignaling in cells expressing human PRLR. As used herein, an antibody orantigen-binding fragment thereof “does not block” prolactin-mediatedsignaling if, when tested in a prolactin signaling blocking assay suchas the assay defined in Example 5 herein or a substantially similarassay, the antibody exhibits no or only negligible blocking activity.According to certain embodiments, an antibody or antigen-bindingfragment “does not block” prolactin-mediated signaling if the antibodyexhibits an IC₅₀ value of greater than about 10 nM, or greater thanabout 100 nM when tested in a prolactin signaling blocking assay such asthe assay defined in Example 5 herein or a substantially similar assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combination thereof.The foregoing list of biological characteristics of the antibodies ofthe invention is not intended to be exhaustive. Other biologicalcharacteristics of the antibodies of the present invention will beevident to a person of ordinary skill in the art from a review of thepresent disclosure including the working Examples herein.

Antibody-Drug Conjugates (ADCs)

The present invention provides antibody-drug conjugates (ADCs)comprising an anti-PRLR antibody or antigen-binding fragment thereofconjugated to a therapeutic moiety such as a cytotoxic agent, achemotherapeutic drug, or a radioisotope.

Cytotoxic agents include any agent that is detrimental to the growth,viability or propagation of cells. Examples of suitable cytotoxic agentsand chemotherapeutic agents that can be conjugated to anti-PRLRantibodies in accordance with this aspect of the invention include,e.g., 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide,1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one,1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine,9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine,anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan,butyric acid, calicheamicins, camptothecin, carminomycins, carmustine,cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide,cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine,diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione,disorazoles, dolastatin (e.g., dolastatin 10), doxorubicin, duocarmycin,echinomycins, eleutherobins, emetine, epothilones, esperamicin,estramustines, ethidium bromide, etoposide, fluorouracils,geldanamycins, gramicidin D, glucocorticoids, irinotecans, kinesinspindle protein (KSP) inhibitors, leptomycins, leurosines, lidocaine,lomustine (CCNU), maytansinoids, mechlorethamine, melphalan,mercatopurines, methopterins, methotrexate, mithramycin, mitomycin,mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine,propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PBDs),rhizoxins, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine,thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine,vincristine, vindesine, vinorelbines, and derivatives of any of theforegoing. According to certain embodiments, the cytotoxic agent that isconjugated to an anti-PRLR antibody is a maytansinoid such as DM1 orDM4, a tomaymycin derivative, or a dolastatin derivative. According tocertain embodiments, the cytotoxic agent that is conjugated to ananti-PRLR antibody is an auristatin such as MMAE, MMAF, or derivativesthereof. Other cytotoxic agents known in the art are contemplated withinthe scope of the present invention, including, e.g., protein toxins suchricin, C. difficile toxin, pseudomonas exotoxin, ricin, diphtheriatoxin, botulinum toxin, bryodin, saporin, pokeweed toxins (i.e.,phytolaccatoxin and phytolaccigenin), and others such as those set forthin Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469.

The present invention also includes antibody-radionuclide conjugates(ARCs) comprising anti-PRLR antibodies conjugated to one or moreradionuclides. Exemplary radionuclides that can be used in the contextof this aspect of the invention include, but are not limited to, e.g.,²²⁵Ac, ²¹²Bi, ²¹³Bi, ¹³¹I, ¹⁸⁶Re, ²²⁷Th, ²²²Rn, ²²³Ra, ²²⁴Ra, and ⁹⁰Y.

In certain embodiments of the present invention, ADCs are providedcomprising an anti-PRLR conjugated to a cytotoxic agent (e.g., any ofthe cytotoxic agents disclosed above) via a linker molecule. Any linkermolecule or linker technology known in the art can be used to create orconstruct an ADC of the present invention. In certain embodiments, thelinker is a cleavable linker. According to other embodiments, the linkeris a non-cleavable linker. Exemplary linkers that can be used in thecontext of the present invention include, linkers that comprise orconsist of e.g., MC (6-maleimidocaproyl), MP (maleimidopropanoyl),val-cit (valine-citrulline), val-ala (valine-alanine), dipeptide site inprotease-cleavable linker, ala-phe (alanine-phenylalanine), dipeptidesite in protease-cleavable linker, PAB (p-am inobenzyloxycarbonyl), SPP(N-Succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate), SIAB (N-Succinimidyl(4-iodo-acetyl)aminobenzoate), and variants and combinations thereof.Additional examples of linkers that can be used in the context of thepresent invention are disclosed, e.g., in U.S. Pat. No. 7,754,681 and inDucry, Bioconjugate Chem., 2010, 21:5-13, and the references citedtherein, the contents of which are incorporated by reference herein intheir entireties.

The present invention comprises ADCs in which a linker connects ananti-PRLR antibody or antigen-binding molecule to a drug or cytotoxinthrough an attachment at a particular amino acid within the antibody orantigen-binding molecule. Exemplary amino acid attachments that can beused in the context of this aspect of the invention include, e.g.,lysine (see, e.g., U.S. Pat. No. 5,208,020; US 2010/0129314; Hollanderet al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808; U.S. Pat.No. 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see,e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873;WO 2013/053872; WO 2011/130598; US 2013/0101546; and U.S. Pat. No.7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al.,Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formyl glycine(see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwalet al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al.,Nat. Protocols, 2012, 10:1052-1067), non-natural amino acids (see, e.g.,WO 2013/068874, and WO 2012/166559), and acidic amino acids (see, e.g.,WO 2012/05982). Linkers can also be conjugated to an antigen-bindingprotein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001,13:127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO2010/010324, WO 2011/018611, and Shaunak et al., Nat. Chem. Biol., 2006,2:312-313).

According to certain embodiments, the present invention provides ADCs,wherein an anti-PRLR antibody as described herein is conjugated to alinker-drug composition as set forth in International Patent ApplicationNo. PCT/US14/29757, filed on Mar. 14, 2014 (e.g., compound “7,” alsoreferred to herein as “M0026”), the disclosure of which is herebyincorporated by reference herein in its entirety.

Any method known in the art for conjugating a chemical moiety to apeptide, polypeptide or other macromolecule can be used in the contextof the present invention to make an anti-PRLR ADC as described herein.An exemplary method for antibody-drug conjugation via a linker is setforth in Example 6 herein. Variations on this exemplary method will beappreciated by persons of ordinary skill in the art and are contemplatedwithin the scope of the present invention.

Targeting ADCs to Cells Expressing Low Levels of PRLR

It was surprisingly discovered by the present inventors that ADCscomprising an anti-PRLR antibody conjugated to a cytotoxic agent areable to specifically target and kill cells that express relatively lowlevels of cell surface PRLR. For example, in Example 7 herein, it isshown that an ADC comprising anti-PRLR antibody H1H6953N conjugated toDM1 was able to inhibit the growth of T47D cells (expressing PRLR atonly 12× above background) with an IC₅₀ of 1.3 nM and showed 78%killing. By contrast, ADCs against other tumor-associated antigens suchas ErbB2 typically require much higher expression levels of the targetantigen on cells for comparable killing potencies. For example, cellkilling in the sub-nanomolar IC₅₀ range was obtained with ananti-ErbB2-DM1 ADC only with cells that express ErbB2 at levels ofgreater than about 200× to about 400× above background (see, e.g.,Tables 14-17 herein). The ability to kill tumor cells that expressrelatively low levels of tumor-associated antigen such as PRLR meansthat the anti-PRLR ADCs of the present invention can provide significanttherapeutic benefits with a lower dose and/or less frequent dosing thanis required for ADCs that target other tumor antigens such as ErbB2.

Accordingly, the present invention provides antibody-drug conjugates(ADCs) comprising an antibody or antigen-binding fragment thereof thatspecifically binds human PRLR conjugated to a cytotoxic agent, whereinthe ADCs effectively kill cells (e.g., tumor cells) that express lowlevels of PRLR. In related embodiments, the present invention includesmethods for effectively killing cells that express low levels of PRLR.The methods according to this aspect of the invention comprisecontacting the cells with an antibody-drug conjugate (ADC) comprising ananti-PRLR antibody conjugated to a cytotoxic agent. “Contacting thecells” can be carried out in vitro, or in vivo, e.g., by administeringan anti-PRLR ADC to a subject in need thereof, wherein theadministration causes the ADC to come into contact with cells expressingPRLR.

According to certain contexts envisioned within the scope of the presentinvention, a “low level of PRLR” means an expression level of less thanabout 30-fold above background. According to certain embodiments,anti-PRLR ADCs are provided which effectively kill cells that expressPRLR at an expression level of less than about 30-fold, 25-fold,20-fold, 18-fold, 16-fold, 14-fold, 12-fold, 10-fold, 8-fold, or less,above background. As used herein, the term “background” means the(non-specific) signal produced when cells are treated with an isotypecontrol antibody (i.e., not specific for PRLR).

In certain other contexts, a “low level of PRLR” can be expressed interms of the number of PRLR molecules per cell. For example, as usedherein, a cell that expresses a “low level” of PRLR expresses less thanabout 1 million copies of PRLR per cell. In specific embodiments, a “lowlevel” of PRLR means less than about 900,000 copies, less than about800,000 copies, less than about 700,000 copies, less than about 600,000copies, less than about 500,000 copies, less than about 400,000 copies,less than about 300,000 copies, less than about 200,000 copies, lessthan about 100,000 copies, less than about 90,000 copies, less thanabout 80,000 copies, less than about 70,000 copies, less than about60,000 copies, less than about 50,000 copies, less than about 40,000copies, less than about 30,000 copies, less than about 20,000 copies, orless than about 10,000 copies of PRLR per cell.

As used herein, “effective killing” means that the ADC exhibits an IC₅₀of less than about 20 nM, or less than about 1 nM (e.g., less than about0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about0.6 nM, less than about 0.5 nM, less than about 0.4 nM, or less thanabout 0.3 nM) in a tumor cell killing assay, such as the assay definedin Example 7 herein, or a substantially similar assay. According to thisaspect of the invention, the anti-PRLR antibody component of the ADC canbe any anti-PRLR antibody including anti-PRLR antibodies comprising anyof the CDR or HCVR/LCVR amino acid sequences as set forth in Table 1herein. Additionally, the cytotoxic agent component of the ADC can beany cytotoxic agent, such as DM1, or any other cytotoxic agent mentionedherein.

ADCs of the present invention are able to inhibit tumor growth and/orreduce tumor size in PRLR+ tumor-bearing animals. For example, as shownin Example 8 herein, anti-PRLR-DM1 ADCs were shown to reduce tumors toundetectable levels in mice bearing PRLR+ breast cancer xenografts.Thus, the present invention includes anti-PRLR antibodies and ADCscomprising such antibodies, wherein the antibodies or ADCs, whenadministered to a PRLR+ tumor-bearing animal (e.g., at a frequency ofabout once a week, and a dose of about 1 to 15 mg/kg), inhibit tumorgrowth and/or reduce tumor size (e.g., tumor growth inhibition of 100%or greater) by Day 52 post-administration or sooner.

Class I Cytokine Receptor Targeting

PRLR belongs to the class I cytokine receptor family. As explained aboveand demonstrated in the working examples herein, it was unexpectedlydiscovered that antibody-drug conjugates (ADCs) comprising anti-PRLRantibodies can effectively target and kill cells that express low levelsof PRLR (see Example 7 herein). Furthermore, it was shown that ADCsagainst other class I cytokine receptors (IL-4R and IL-6R) also are ableto potently kill cell lines expressing relatively low levels of targetantigen (see Example 9 herein). This property is in contrast to ADCsagainst other cell surface-expressed proteins, such as ErbB2, whereineffective cell killing requires high target expression. Moreover, it wasalso surprisingly discovered that anti-PRLR antibodies are internalizedsubstantially faster than anti-Her2 antibodies on tumor cells (e.g.,T47D tumor cells), and that this property is correlated with fasterinternalization and degradation of cell surface PRLR compared to cellsurface Her2.

In view of the results set forth herein, the present inventors conceivedthat the ability to target and kill cells that express low levels ofcell surface antigen may be a common property shared by ADCs directedagainst class I cytokine receptors in general, and in particular class Icytokine receptors that are rapidly internalized. Thus, the presentinvention includes methods for targeting class I cytokine receptors(e.g., rapidly internalizing class I cytokine receptors), and methodsfor killing cells that express class I cytokine receptors such as cellsthat express low levels of class I cytokine receptors.

The methods according to this aspect of the invention comprisecontacting a cell that expresses a class I cytokine receptor with an ADCcomprising an antibody or antigen-binding fragment thereof thatspecifically binds the class I cytokine receptor. According to certainembodiments, the cell to be targeted expresses low levels (as thatexpression is defined elsewhere herein) of a class I cytokine receptorand/or a class I cytokine receptor that is rapidly internalized anddegraded (e.g., internalized faster than a reference cell surfacemolecule such as Her2). Also included within the present invention areADCs comprising an antibody or antigen-binding fragment thereof thatspecifically binds a class I cytokine receptor, conjugated to acytotoxic agent. Any of the cytotoxic agents, linkers, and/orADC-related technologies described elsewhere herein can be used in thecontext of this aspect of the invention.

As used herein a “class I cytokine receptor” (also sometimes referred toas a “type I cytokine receptor”) means a transmembrane receptorexpressed on the surface of cells that recognizes and responds tocytokines with four alpha-helical strands. As explained below, class Icytokine receptors can be heterodimeric or homodimeric. As used herein,the term “class I cytokine receptor” includes both heterodimeric andhomodimeric receptors.

Heterodimeric class I cytokine receptors consist of a cytokine-specificchain and a “common chain.” Accordingly, such heterodimeric class Icytokine receptors can be classified based on the type of common chainused by the receptor for signaling. Exemplary categories ofheterodimeric class I cytokine receptors include: (i) common gammachain-containing heterodimeric receptors such as IL-2R, IL-4R, IL-7R,IL-9R, IL-13R and IL-15R; (ii) common beta chain-containingheterodimeric receptors such as GM-CSF receptor, IL-3R and IL-5R; and(iii) gp130-containing heterodimeric receptors such as IL-6R, IL-11R,CNTF receptor, leukemia inhibitory factor (LIF) receptor, oncostatin M(OSM) receptor, and IL-12 receptor.

Homodimeric class I cytokine receptors include growth hormone (GH)receptor, erythropoietin (EPO) receptor, G-CSF receptor, leptinreceptor, and PRLR.

In certain embodiments of this aspect of the invention, the ADCcomprises an antibody or antigen-binding fragment thereof thatspecifically binds a heterodimeric class I cytokine receptor. Accordingto other embodiments of this aspect of the invention, the ADC comprisesan antibody or antigen-binding fragment thereof that specifically bindsa homodimeric class I cytokine receptor.

The present invention includes methods for killing a cell that expresseslow levels of a heterodimeric class I cytokine receptor. The methodsaccording to this aspect of the invention comprise contacting a cellthat expresses a low level of a heterodimeric class I cytokine receptorwith an ADC comprising an antibody or antigen-binding fragment thereofthat specifically binds the heterodimeric class I cytokine receptor.

Alternatively, the present invention includes methods for killing a cellthat expresses low levels of a homodimeric class I cytokine receptor.The methods according to this aspect of the invention comprisecontacting a cell that expresses a low level of a homodimeric class Icytokine receptor with an ADC comprising an antibody or antigen-bindingfragment thereof that specifically binds the homodimeric class Icytokine receptor.

Epitope Mapping and Related Technologies

The epitope to which the antibodies of the present invention bind mayconsist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acidsof a PRLR protein. Alternatively, the epitope may consist of a pluralityof non-contiguous amino acids (or amino acid sequences) of PRLR. In someembodiments, the epitope is located on or near the prolactin-bindingdomain of PRLR. In other embodiments, the epitope is located outside ofthe prolactin-binding domain of PRLR, e.g., at a location on the surfaceof PRLR at which an antibody, when bound to such an epitope, does notinterfere with prolactin binding to PRLR.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-PRLR antibodies that bind tothe same epitope as any of the specific exemplary antibodies describedherein (e.g. antibodies comprising any of the amino acid sequences asset forth in Table 1 herein). Likewise, the present invention alsoincludes anti-PRLR antibodies that compete for binding to PRLR with anyof the specific exemplary antibodies described herein (e.g. antibodiescomprising any of the amino acid sequences as set forth in Table 1herein).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-PRLR antibody byusing routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-PRLR antibody of the invention, the reference antibody isallowed to bind to a PRLR protein. Next, the ability of a test antibodyto bind to the PRLR molecule is assessed. If the test antibody is ableto bind to PRLR following saturation binding with the referenceanti-PRLR antibody, it can be concluded that the test antibody binds toa different epitope than the reference anti-PRLR antibody. On the otherhand, if the test antibody is not able to bind to the PRLR moleculefollowing saturation binding with the reference anti-PRLR antibody, thenthe test antibody may bind to the same epitope as the epitope bound bythe reference anti-PRLR antibody of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, Biacore, flow cytometry or any otherquantitative or qualitative antibody-binding assay available in the art.In accordance with certain embodiments of the present invention, twoantibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-,10-, 20- or 100-fold excess of one antibody inhibits binding of theother by at least 50% but preferably 75%, 90% or even 99% as measured ina competitive binding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antibodies are deemed to bind tothe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-PRLR antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to a PRLR proteinunder saturating conditions followed by assessment of binding of thetest antibody to the PRLR molecule. In a second orientation, the testantibody is allowed to bind to a PRLR molecule under saturatingconditions followed by assessment of binding of the reference antibodyto the PRLR molecule. If, in both orientations, only the first(saturating) antibody is capable of binding to the PRLR molecule, thenit is concluded that the test antibody and the reference antibodycompete for binding to PRLR. As will be appreciated by a person ofordinary skill in the art, an antibody that competes for binding with areference antibody may not necessarily bind to the same epitope as thereference antibody, but may sterically block binding of the referenceantibody by binding an overlapping or adjacent epitope.

Preparation of Human Antibodies

The anti-PRLR antibodies of the present invention can be fully humanantibodies. Methods for generating monoclonal antibodies, includingfully human monoclonal antibodies are known in the art. Any such knownmethods can be used in the context of the present invention to makehuman antibodies that specifically bind to human PRLR.

Using VELOCIMMUNE™ technology, for example, or any other similar knownmethod for generating fully human monoclonal antibodies, high affinitychimeric antibodies to PRLR are initially isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, ligand blocking activity,selectivity, epitope, etc. If necessary, mouse constant regions arereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4, to generate a fully human anti-PRLR antibody.While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region. In certain instances, fully humananti-PRLR antibodies are isolated directly from antigen-positive Bcells.

Bioequivalents

The anti-PRLR antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies but that retain the ability to bind human PRLR.Such variant antibodies and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-PRLRantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-PRLR antibody or antibody fragment that isessentially bioequivalent to an anti-PRLR antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-PRLR antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-PRLR antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesanti-PRLR antibodies that bind to human PRLR but not to PRLR from otherspecies. The present invention also includes anti-PRLR antibodies thatbind to human PRLR and to PRLR from one or more non-human species. Forexample, the anti-PRLR antibodies of the invention may bind to humanPRLR and may bind or not bind, as the case may be, to one or more ofmouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzeePRLR. According to certain exemplary embodiments of the presentinvention, anti-PRLR antibodies are provided which specifically bindhuman PRLR and cynomolgus monkey (e.g., Macaca fascicularis) PRLR. Otheranti-PRLR antibodies of the invention bind human PRLR but do not bind,or bind only weakly, to cynomolgus monkey PRLR.

Multispecific Antibodies

The antibodies of the present invention may be monospecific ormultispecific (e.g., bispecific). Multispecific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-PRLR antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity.

The present invention includes bispecific antibodies wherein one arm ofan immunoglobulin binds human PRLR, and the other arm of theimmunoglobulin is specific for a second antigen. The PRLR-binding armcan comprise any of the HCVR/LCVR or CDR amino acid sequences as setforth in Table 1 herein. In certain embodiments, the PRLR-binding armbinds human PRLR and blocks prolactin binding to PRLR. In otherembodiments, the PRLR-binding arm binds human PRLR but does not blockprolactin binding to PRLR.

An exemplary bispecific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bispecificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-PRLR antibodies or antigen-binding fragments thereof of the presentinvention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. In an adultpatient, it may be advantageous to intravenously administer the antibodyof the present invention normally at a single dose of about 0.01 toabout 20 mg/kg body weight, more preferably about 0.02 to about 7, about0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Dependingon the severity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering anti-PRLR antibodies may be determined empirically; forexample, patient progress can be monitored by periodic assessment, andthe dose adjusted accordingly. Moreover, interspecies scaling of dosagescan be performed using well-known methods in the art (e.g., Mordenti etal., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-PRLR antibody or an antibody-drug conjugate comprising an anti-PRLRantibody (e.g., an anti-PRLR antibody or ADC comprising any of theHCVR/LCVR or CDR sequences as set forth in Table 1 herein). Thetherapeutic composition can comprise any of the anti-PRLR antibodies,antigen-binding fragments thereof, or ADCs disclosed herein, and apharmaceutically acceptable carrier or diluent.

The antibodies and ADCs of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by PRLR expression or activity, or treatableby blocking the interaction between PRLR and prolactin or otherwiseinhibiting PRLR activity and/or signaling, and/or promoting receptorinternalization and/or decreasing cell surface receptor number. Forexample, the antibodies and ADCs of the present invention are useful forthe treatment of tumors that express PRLR and/or that respond toprolactin-mediated signaling, e.g., breast tumors. The antibodies andantigen-binding fragments of the present invention may also be used totreat primary and/or metastatic tumors arising in the brain andmeninges, oropharynx, lung and bronchial tree, gastrointestinal tract,male and female reproductive tract, muscle, bone, skin and appendages,connective tissue, spleen, immune system, blood forming cells and bonemarrow, liver and urinary tract, and special sensory organs such as theeye. In certain embodiments, the antibodies and ADCs of the inventionare used to treat one or more of the following cancers: renal cellcarcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer,malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer(e.g., gastric cancer with MET amplification), malignant mesothelioma,multiple myeloma, ovarian cancer, small cell lung cancer, non-small celllung cancer, synovial sarcoma, thyroid cancer, breast cancer, ormelanoma.

The anti-PRLR antibodies of the present invention are also useful forthe treatment or prevention of one or more diseases or disordersselected from the group consisting of endometriosis, adenomyosis,non-hormonal female fertility contraception, benign breast disease andmastalgia, lactation inhibition, benign prostate hyperplasia, fibroids,hyper- and normoprolactinemic hair loss, and as part of hormone therapyto inhibit mammary epithelial cell proliferation.

In the context of the methods of treatment described herein, theanti-PRLR antibody may be administered as a monotherapy (i.e., as theonly therapeutic agent) or in combination with one or more additionaltherapeutic agents (examples of which are described elsewhere herein).

The present invention includes methods for identifying patients who aretreatable with an antibody or ADC of the present invention by assayingfor high levels of PRLR expression in one or more tissues of the patientsuch as a tumor tissue. In a related embodiment, the present inventionincludes methods for treating cancers characterized by high levelexpression of PRLR. For example, the present invention includes methodsof treatment comprising administering an anti-PRLR antibody of theinvention, or ADC thereof (e.g., any of the anti-PRLR ADCs describedelsewhere herein), to a subject with a tumor, wherein the tumor has beenidentified as expressing high levels of PRLR. In certain embodiments,the tumor is identified as expressing high levels of PRLR byimmunohistochemistry of a biopsy sample or other imaging techniques suchas, e.g., immuno-PET imaging, etc.

Combination Therapies and Formulations

The present invention includes compositions and therapeutic formulationscomprising any of the anti-PRLR antibodies described herein incombination with one or more additional therapeutically activecomponents, and methods of treatment comprising administering suchcombinations to subjects in need thereof.

The anti-PRLR antibodies of the present invention may be co-formulatedwith and/or administered in combination with one or more additionaltherapeutically active component(s) selected from the group consistingof: an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab orpanitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib orerlotinib]), an antagonist of another EGFR family member such asHer2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1{KADCYLA®}], anti-ErbB3 or anti-ErbB4 antibody or small moleculeinhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII(e.g., an antibody that specifically binds EGFRvIII), a cMET anagonist(e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1Rantibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879,PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), aPDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody or small moleculekinase inhibitor such as, e.g., imatinib mesylate or sunitinib malate),a PDGF ligand inhibitor (e.g., anti-PDGF-A, -B, -C, or -D antibody,aptamer, siRNA, etc.), a VEGF antagonist (e.g., a VEGF-Trap such asaflibercept, see, e.g., U.S. Pat. No. 7,087,411 (also referred to hereinas a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), a FOLH1 antagonist (e.g., an anti-FOLH1antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibodyor an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g.,an anti-uroplakin [e.g., anti-UPK3A] antibody), a MUC16 antagonist(e.g., an anti-MUC16 antibody), a Tn antigen antagonist (e.g., ananti-Tn antibody), a CLEC12A antagonist (e.g., an anti-CLEC12Aantibody), a TNFRSF17 antagonist (e.g., an anti-TNFRSF17 antibody), aLGRS antagonist (e.g., an anti-LGRS antibody), a monovalent CD20antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab),etc. Other agents that may be beneficially administered in combinationwith antibodies of the invention include, e.g., tamoxifen, aromataseinhibitors, and cytokine inhibitors, including small-molecule cytokineinhibitors and antibodies that bind to cytokines such as IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18,or to their respective receptors.

The present invention includes compositions and therapeutic formulationscomprising any of the anti-PRLR antibodies described herein incombination with one or more chemotherapeutic agents. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide (Cytoxan™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (Taxotere™; Aventis Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The anti-PRLR antibodies of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids, steroids, oxygen, antioxidants, COXinhibitors, cardioprotectants, metal chelators, IFN-gamma, and/orNSAIDs.

The additional therapeutically active component(s), e.g., any of theagents listed above or derivatives thereof, may be administered justprior to, concurrent with, or shortly after the administration of ananti-PRLR antibody of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-PRLR antibody “in combination with” anadditional therapeutically active component). The present inventionincludes pharmaceutical compositions in which an anti-PRLR antibody ofthe present invention is co-formulated with one or more of theadditional therapeutically active component(s) as described elsewhereherein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-PRLR antibody (or a pharmaceutical compositioncomprising a combination of an anti-PRLR antibody and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-PRLR antibody ofthe invention. As used herein, “sequentially administering” means thateach dose of anti-PRLR antibody is administered to the subject at adifferent point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of an anti-PRLR antibody, followed byone or more secondary doses of the anti-PRLR antibody, and optionallyfollowed by one or more tertiary doses of the anti-PRLR antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-PRLR antibody ofthe invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-PRLR antibody, but generally may differ from one another in termsof frequency of administration. In certain embodiments, however, theamount of anti-PRLR antibody contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1%, 2,2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%,12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%,20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-PRLR antibody which is administered toa patient prior to the administration of the very next dose in thesequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PRLR antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient. The administration regimen may becarried out indefinitely over the lifetime of a particular subject, oruntil such treatment is no longer therapeutically needed oradvantageous.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient at a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of once a month, then the maintenance dosesmay be administered to the patient once every six weeks, once every twomonths, once every three months, etc.

Diagnostic Uses of the Antibodies

The anti-PRLR antibodies of the present invention may also be used todetect and/or measure PRLR, or PRLR-expressing cells in a sample, e.g.,for diagnostic purposes. For example, an anti-PRLR antibody, or fragmentthereof, may be used to diagnose a condition or disease characterized byaberrant expression (e.g., over-expression, under-expression, lack ofexpression, etc.) of PRLR. Exemplary diagnostic assays for PRLR maycomprise, e.g., contacting a sample, obtained from a patient, with ananti-PRLR antibody of the invention, wherein the anti-PRLR antibody islabeled with a detectable label or reporter molecule. Alternatively, anunlabeled anti-PRLR antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein, or rhodamine; or an enzymesuch as alkaline phosphatase, beta-galactosidase, horseradishperoxidase, or luciferase. Specific exemplary assays that can be used todetect or measure PRLR in a sample include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA), immuno-PET (e.g., ⁸⁹Zr, ⁶⁴Cu,etc.), and fluorescence-activated cell sorting (FACS).

Samples that can be used in PRLR diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of PRLR protein, orfragments thereof, under normal or pathological conditions. Generally,levels of PRLR in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal PRLR levels or activity) will be measured to initiallyestablish a baseline, or standard, level of PRLR. This baseline level ofPRLR can then be compared against the levels of PRLR measured in samplesobtained from individuals suspected of having a PRLR related disease orcondition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Anti-PRLR Antibodies

Anti-PRLR antibodies were obtained by immunizing a VELOCIMMUNE® mouse(i.e., an engineered mouse comprising DNA encoding human immunoglobulinheavy and kappa light chain variable regions) with an immunogencomprising a soluble dimeric ecto domain of human PRLR. The antibodyimmune response was monitored by a PRLR-specific immunoassay. When adesired immune response was achieved splenocytes were harvested andfused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines were screened andselected to identify cell lines that produce PRLR-specific antibodies.Using this technique several anti-PRLR chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained. In addition, several fully human anti-PRLR antibodieswere isolated directly from antigen-positive B cells without fusion tomyeloma cells, as described in US 2007/0280945A1.

Certain biological properties of the exemplary anti-PRLR antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-PRLR antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H6762P 2 4 6 8 10 12 1416 H1H6765P 18 20 22 24 26 28 30 32 H1H6774P 34 36 38 40 42 44 46 48H1H6781P 50 52 54 56 58 60 62 64 H1H6782P 66 68 70 72 74 76 78 80H1H6783P 82 84 86 88 90 92 94 96 H1H6785P 98 100 102 104 106 108 110 112H1H6790P 114 116 118 120 122 124 126 128 H1H6792P 130 132 134 136 138140 142 144 H1H6793P 146 148 150 152 154 156 158 160 H1H6795P 162 164166 168 170 172 174 176 H1H6797P 178 180 182 184 186 188 190 192H1H6800P 194 196 198 200 202 204 206 208 H1H6801P 210 212 214 216 218220 222 224 H1H6803P 226 228 230 232 234 236 238 240 H1H6804P 242 244246 248 250 252 254 256 H1H6807P 258 260 262 264 266 268 270 272H1M6953N 274 276 278 280 282 284 286 288 H2M6958N2 290 292 294 296 298300 302 304 H2M6959N2 306 308 310 312 314 316 318 320 H2M6960N 322 324326 328 330 332 334 336 H2M6966N 338 340 342 344 346 348 350 352H2M6967N 354 356 358 360 362 364 366 368 H2M6975N 370 372 374 376 378380 382 384 H2M6976N 386 388 390 392 394 396 398 400

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H6762P 1 3 57 9 11 13 15 H1H6765P 17 19 21 23 25 27 29 31 H1H6774P 33 35 37 39 41 4345 47 H1H6781P 49 51 53 55 57 59 61 63 H1H6782P 65 67 69 71 73 75 77 79H1H6783P 81 83 85 87 89 91 93 95 H1H6785P 97 99 101 103 105 107 109 111H1H6790P 113 115 117 119 121 123 125 127 H1H6792P 129 131 133 135 137139 141 143 H1H6793P 145 147 149 151 153 155 157 159 H1H6795P 161 163165 167 169 171 173 175 H1H6797P 177 179 181 183 185 187 189 191H1H6800P 193 195 197 199 201 203 205 207 H1H6801P 209 211 213 215 217219 221 223 H1H6803P 225 227 229 231 233 235 237 239 H1H6804P 241 243245 247 249 251 253 255 H1H6807P 257 259 261 263 265 267 269 271H1M6953N 273 275 277 279 281 283 285 287 H2M6958N2 289 291 293 295 297299 301 303 H2M6959N2 305 307 309 311 313 315 317 319 H2M6960N 321 323325 327 329 331 333 335 H2M6966N 337 339 341 343 345 347 349 351H2M6967N 353 355 357 359 361 363 365 367 H2M6975N 369 371 373 375 377379 381 383 H2M6976N 385 387 389 391 393 395 397 399

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “HIM,” “H2M,” etc.), followed by anumerical identifier (e.g. “6762,” “6953,” “6958,” etc.), followed by a“P” or “N” suffix, as shown in Tables 1 and 2. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H1H6762P,” “H1M6953N,” “H2M6958N,” etc. The H1H, H1M and H2M prefixeson the antibody designations used herein indicate the particular Fcregion isotype of the antibody. For example, an “H1H” antibody has ahuman IgG1 Fc, an “HIM” antibody has a mouse IgG1 Fc, and an “H2M”antibody has a mouse IgG2 Fc, (all variable regions are fully human asdenoted by the first ‘H’ in the antibody designation). As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Tables 1 and 2—will remain the same, and thebinding properties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

Control Constructs Used in the Following Examples

Control constructs were included in the following experiments forcomparative purposes: Control I: a human anti-PRLR antibody with heavyand light chain variable domains having the amino acid sequences of thecorresponding domains of “he.06.642-2,” as set forth in WO2008/02295A2;and Control II: a human anti-ErbB2 antibody with heavy and light chainvariable domains having the amino acid sequences of the correspondingdomains of “4D5v8” as set forth in: Carter et al., 1992, Proc. Natl.Acad. Sci. USA, 89:4285-4289.

Example 3. Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-PRLR Antibodies

Binding affinities and kinetic constants of human monoclonal anti-PRLRantibodies were determined by surface plasmon resonance at 25° C. and37° C. Antibodies, expressed as human IgG1 Fc “H1H” designations), werecaptured onto an anti-human Fc sensor surface (mAb-capture format), andsoluble monomeric (hPRLR.mmh; SEQ ID NO:401, or macaca fascicularis (mf)PRLR.mmh; SEQ ID NO:403) or dimeric (hPRLR.mFc; SEQ ID NO:402) PRLRprotein was injected over the sensor surface. Measurements wereconducted on a T200 Biacore instrument. Kinetic association (k_(a)) anddissociation (k_(d)) rate constants were determined by processing andfitting the data to a 1:1 binding model using Scrubber 2.0 curve fittingsoftware. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (t_(1/2)) were calculated from the kinetic rateconstants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min)=(ln 2/(60*k_(d)).Results are summarized in Tables 3 and 4.

TABLE 3 Biacore Binding Affinities of Human Fc mAbs at 25° C. Binding at25° C./Antibody-Capture Format ka kd K_(D) T½ Antibody Analyte (Ms⁻¹)(s⁻¹) (Molar) (min) H1H6953N hPRLR.mmh 7.92E+05 2.12E−04 2.68E−10 54.5hPRLR.mFc 7.11E+05 4.77E−05 6.70E−11 242.2 mfPRLR.mmh 7.27E+05 3.19E−044.38E−10 36.3 H1H6958N2 hPRLR.mmh 2.33E+05 2.72E−04 1.17E−09 42.5hPRLR.mFc 4.22E+05 3.66E−05 8.67E−11 315.8 mfPRLR.mmh 1.95E+05 2.80E−041.44E−09 41.3 H1H6959N2 hPRLR.mmh 1.79E+05 3.60E−02 2.02E−07 0.3hPRLR.mFc 3.59E+05 5.18E−04 1.45E−09 22.3 mfPRLR.mmh 1.29E+05 1.67E−021.29E−07 0.7 H1H6960N hPRLR.mmh 1.08E+05 7.29E−04 6.74E−09 15.8hPRLR.mFc 2.52E+05 3.56E−05 1.41E−10 324.4 mfPRLR.mmh 9.40E+04 5.83E−046.20E−09 19.8 H1H6966N hPRLR.mmh 8.52E+05 2.57E−04 3.02E−10 44.9hPRLR.mFc 9.31E+05 4.08E−05 4.39E−11 282.9 mfPRLR.mmh 7.82E+05 3.25E−044.16E−10 35.6 H1H6967N hPRLR.mmh 2.46E+05 3.32E−04 1.35E−09 34.8hPRLR.mFc 4.07E+05 4.45E−05 1.10E−10 259.3 mfPRLR.mmh 1.90E+05 5.85E−043.08E−09 19.7 H1H6975N hPRLR.mmh 1.50E+05 7.35E−05 4.90E−10 157.1hPRLR.mFc 2.87E+05 1.97E−05 6.84E−11 587.8 mfPRLR.mmh 1.15E+05 1.31E−041.14E−09 88.2 H1H6976N hPRLR.mmh 5.44E+05 8.64E−04 1.59E−09 13.4hPRLR.mFc 1.12E+06 1.01E−04 9.06E−11 114.4 mfPRLR.mmh 4.66E+05 8.14E−041.75E−09 14.2 H1H6762P hPRLR.mmh 6.92E+05 1.79E−04 2.59E−10 64 hPRLR.mFc6.51E+05 6.45E−05 9.92E−11 179 mfPRLR.mmh 4.08E+05 2.08E−04 5.11E−10 55H1H6765P hPRLR.mmh 9.07E+05 1.13E−04 1.24E−10 102 hPRLR.mFc 8.69E+053.41E−05 3.92E−11 339 mfPRLR.mmh 5.27E+05 1.34E−04 2.54E−10 86 H1H6774PhPRLR.mmh 7.15E+05 8.18E−04 1.14E−09 14 hPRLR.mFc 7.98E+05 8.94E−051.12E−10 129 mfPRLR.mmh 4.95E+05 8.75E−04 1.77E−09 13 H1H6781P hPRLR.mmh2.08E+05 1.27E−04 6.10E−10 91 hPRLR.mFc 2.57E+05 6.05E−05 2.36E−10 191mfPRLR.mmh 1.43E+05 1.41E−04 9.86E−10 82 H1H6782P hPRLR.mmh 3.60E+052.47E−04 6.85E−10 47 hPRLR.mFc 3.17E+05 7.13E−05 2.25E−10 162 mfPRLR.mmh2.66E+05 2.79E−04 1.05E−09 41 H1H6783P hPRLR.mmh 2.88E+05 4.13E−041.43E−09 28 hPRLR.mFc 2.64E+05 8.77E−05 3.32E−10 132 mfPRLR.mmh 1.89E+053.41E−04 1.81E−09 34 H1H6785P hPRLR.mmh 3.01E+05 1.71E−04 5.67E−10 68hPRLR.mFc 2.63E+05 6.07E−05 2.31E−10 190 mfPRLR.mmh 2.27E+05 1.67E−047.38E−10 69 H1H6790P hPRLR.mmh 5.65E+05 7.99E−04 1.41E−09 14 hPRLR.mFc5.42E+05 1.04E−04 1.92E−10 111 mfPRLR.mmh 3.89E+05 7.93E−04 2.04E−09 15H1H6792P hPRLR.mmh 3.24E+05 7.94E−04 2.45E−09 15 hPRLR.mFc 3.03E+059.48E−05 3.13E−10 122 mfPRLR.mmh 2.36E+05 9.83E−04 4.17E−09 12 H1H6793PhPRLR.mmh 2.35E+05 3.32E−04 1.41E−09 35 hPRLR.mFc 2.29E+05 7.57E−053.31E−10 153 mfPRLR.mmh 1.77E+05 3.93E−04 2.22E−09 29 H1H6795P hPRLR.mmh1.17E+06 1.77E−03 1.52E−09 7 hPRLR.mFc 1.54E+06 8.41E−05 5.45E−11 137mfPRLR.mmh 8.44E+05 1.97E−03 2.33E−09 6 H1H6797P hPRLR.mmh 1.13E+061.96E−03 1.73E−09 6 hPRLR.mFc 9.82E+05 1.19E−04 1.22E−10 97 mfPRLR.mmh6.70E+05 2.09E−03 3.12E−09 6 H1H6800P hPRLR.mmh 4.21E+05 4.09E−049.72E−10 28 hPRLR.mFc 4.73E+05 8.69E−05 1.84E−10 133 mfPRLR.mmh 3.03E+053.80E−04 1.25E−09 30 H1H6801P hPRLR.mmh 8.46E+05 7.56E−04 8.94E−10 15hPRLR.mFc 6.75E+05 1.09E−04 1.61E−10 106 mfPRLR.mmh 6.57E+05 1.23E−031.88E−09 9 H1H6803P hPRLR.mmh 8.24E+04 1.37E−04 1.67E−09 84 hPRLR.mFc1.04E+05 6.29E−05 6.07E−10 184 mfPRLR.mmh 6.21E+04 2.09E−04 3.37E−09 55H1H6804P hPRLR.mmh 4.53E+05 6.34E−04 1.40E−09 18 hPRLR.mFc 4.51E+058.69E−05 1.93E−10 133 mfPRLR.mmh 3.31E+05 6.57E−04 1.99E−09 18 H1H6807PhPRLR.mmh 7.61E+05 1.44E−04 1.89E−10 80 hPRLR.mFc 6.80E+05 5.46E−058.03E−11 212 mfPRLR.mmh 4.37E+05 1.51E−04 3.46E−10 76 Control IhPRLR.mmh 5.11E+05 7.44E−04 1.46E−09 15.5 hPRLR.mFc 4.72E+05 7.53E−051.59E−10 153.5 mfPRLR.mmh 2.38E+05 6.14E−03 2.59E−08 1.9 NB = No bindingobserved under conditions used

TABLE 4 Biacore Binding Affinities of Human Fc mAbs at 37° C. Binding at37° C./Antibody-Capture Format ka kd K_(D) T½ Antibody Analyte (Ms⁻¹)(s⁻¹) (Molar) (min) H1H6953N hPRLR.mmh 1.10E+06 1.22E−03 1.11E−09 9.4hPRLR.mFc 1.47E+06 1.70E−04 1.16E−10 68.0 mfPRLR.mmh 9.47E+05 2.56E−032.71E−09 4.5 H1H6958N2 hPRLR.mmh 4.13E+05 1.31E−03 3.16E−09 8.8hPRLR.mFc 8.29E+05 1.39E−04 1.67E−10 83.3 mfPRLR.mmh 3.28E+05 1.34E−034.08E−09 8.6 H1H6959N2 hPRLR.mmh 4.06E+04 2.77E−02 6.81E−07 0.4hPRLR.mFc 5.09E+05 2.30E−03 4.51E−09 5.0 mfPRLR.mmh 4.46E+04 1.65E−023.70E−07 0.7 H1H6960N hPRLR.mmh 1.22E+05 1.98E−03 1.62E−08 5.8 hPRLR.mFc2.94E+05 1.47E−04 5.00E−10 78.7 mfPRLR.mmh 8.64E+04 1.28E−03 1.49E−089.0 H1H6966N hPRLR.mmh 1.58E+06 9.60E−04 6.07E−10 12.0 hPRLR.mFc1.88E+06 1.27E−04 6.72E−11 91.3 mfPRLR.mmh 1.22E+06 1.20E−03 9.82E−109.6 H1H6967N hPRLR.mmh 4.24E+05 9.33E−04 2.20E−09 12.4 hPRLR.mFc9.07E+05 9.73E−05 1.07E−10 118.8 mfPRLR.mmh 3.56E+05 1.62E−03 4.54E−097.1 H1H6975N hPRLR.mmh 2.11E+05 2.73E−04 1.29E−09 42.3 hPRLR.mFc3.86E+05 7.09E−05 1.84E−10 163.0 mfPRLR.mmh 1.40E+05 3.17E−04 2.27E−0936.4 H1H6976N hPRLR.mmh 7.77E+05 3.14E−03 4.04E−09 3.7 hPRLR.mFc1.37E+06 1.40E−04 1.02E−10 82.6 mfPRLR.mmh 6.03E+05 3.16E−03 5.24E−093.7 H1H6762P hPRLR.mmh 9.48E+05 4.55E−04 4.80E−10 25 hPRLR.mFc 8.01E+051.03E−04 1.28E−10 112 mfPRLR.mmh 6.79E+05 5.58E−04 8.23E−10 21 H1H6765PhPRLR.mmh 1.25E+06 3.66E−04 2.92E−10 32 hPRLR.mFc 8.01E+05 1.03E−041.28E−10 112 mfPRLR.mmh 1.06E+06 5.37E−05 5.04E−11 215 H1H6774PhPRLR.mmh 1.07E+06 3.17E−03 2.95E−09 4 hPRLR.mFc 1.41E+06 1.94E−041.38E−10 60 mfPRLR.mmh 7.23E+05 3.61E−03 5.00E−09 3 H1H6781P hPRLR.mmh3.39E+05 3.09E−04 9.10E−10 37 hPRLR.mFc 4.36E+05 9.84E−05 2.26E−10 117mfPRLR.mmh 2.47E+05 2.67E−04 1.08E−09 43 H1H6782P hPRLR.mmh 5.57E+057.16E−04 1.28E−09 16 hPRLR.mFc 5.98E+05 1.30E−04 2.17E−10 89 mfPRLR.mmh3.85E+05 7.67E−04 1.99E−09 15 H1H6783P hPRLR.mmh 4.11E+05 1.61E−033.91E−09 7 hPRLR.mFc 4.91E+05 1.38E−04 2.82E−10 83 mfPRLR.mmh 2.73E+051.30E−03 4.74E−09 9 H1H6785P hPRLR.mmh 4.26E+05 4.84E−04 1.14E−09 24hPRLR.mFc 4.56E+05 1.17E−04 2.56E−10 99 mfPRLR.mmh 2.97E+05 4.50E−041.52E−09 26 H1H6790P hPRLR.mmh 9.40E+05 3.29E−03 3.50E−09 4 hPRLR.mFc6.46E+05 1.98E−04 3.06E−10 58 mfPRLR.mmh 6.15E+05 3.21E−03 5.22E−09 4H1H6792P hPRLR.mmh 4.35E+05 2.29E−03 5.27E−09 5 hPRLR.mFc 4.99E+051.76E−04 3.52E−10 66 mfPRLR.mmh 3.05E+05 2.86E−03 9.37E−09 4 H1H6793PhPRLR.mmh 3.39E+05 1.02E−03 3.02E−09 11 hPRLR.mFc 4.10E+05 1.42E−043.47E−10 81 mfPRLR.mmh 2.33E+05 1.07E−03 4.59E−09 11 H1H6795P hPRLR.mmh1.36E+06 5.20E−03 3.81E−09 2 hPRLR.mFc 1.94E+06 7.77E−05 4.02E−11 149mfPRLR.mmh 9.73E+05 5.99E−03 6.16E−09 2 H1H6797P hPRLR.mmh 1.29E+068.22E−03 6.37E−09 1 hPRLR.mFc 1.80E+06 1.25E−04 6.91E−11 93 mfPRLR.mmh9.14E+05 9.06E−03 9.90E−09 1 H1H6800P hPRLR.mmh 8.08E+05 1.19E−031.47E−09 10 hPRLR.mFc 6.44E+05 1.47E−04 2.29E−10 79 mfPRLR.mmh 4.39E+051.09E−03 2.48E−09 11 H1H6801P hPRLR.mmh 9.51E+05 4.41E−03 4.63E−09 3hPRLR.mFc 7.93E+05 2.21E−04 2.79E−10 52 mfPRLR.mmh 7.11E+05 7.71E−031.08E−08 1 H1H6803P hPRLR.mmh 1.29E+05 3.64E−04 2.83E−09 32 hPRLR.mFc1.34E+05 6.20E−05 4.61E−10 186 mfPRLR.mmh 8.73E+04 6.36E−04 7.28E−09 18H1H6804P hPRLR.mmh 6.07E+05 3.85E−03 6.34E−09 3 hPRLR.mFc 5.54E+052.05E−04 3.69E−10 56 mfPRLR.mmh 4.55E+05 4.26E−03 9.35E−09 3 H1H6807PhPRLR.mmh 1.08E+06 3.36E−04 3.10E−10 34 hPRLR.mFc 1.22E+06 1.10E−049.00E−11 105 mfPRLR.mmh 8.02E+05 3.59E−04 4.48E−10 32 Control IhPRLR.mmh 5.99E+05 2.42E−03 4.04E−09 4.8 hPRLR.mFc 8.47E+05 2.14E−042.53E−10 54.0 mfPRLR.mmh 1.53E+05 1.54E−02 1.01E−07 0.8 NB = No bindingobserved under conditions used

As shown in Tables 3 and 4, several antibodies of the inventiondisplayed sub-nanomolar affinities to human and monkey PRLR protein andexhibited higher affinity than the comparator anti-PRLR antibody(Control I). For example, at 37° C., many of the anti-PRLR antibodies ofthe invention bound to monomeric human PRLR with K_(D) values less than4 nM and T % times greater than 5 minutes; and to dimeric human PRLRwith K_(D) values less than 250 pM and T % times greater than 60minutes. These binding characteristics are substantially better thanwhat was observed with the Control I antibody under the sameexperimental conditions.

Example 4A. Anti-PRLR Antibodies Bind to Endogenous and OverexpressedPRLR Cell Lines

The ability of anti-PRLR antibodies to selectively bind PRLR expressingcell lines was next determined. Human, monkey macaca fascicularis, andmouse PRLR constructs with an HA tag were stably introduced into HEK293cells via Lipofectamine 2000-mediated transfection methodology.Transfectants (HEK293/hPRLR, HEK293/mfPRLR and HEK293/mPRLR) wereselected for at least 2 weeks in complete growth media plus G418.

Cell surface expression of PRLR on 293/hPRLR cells was assessed via FACSanalysis. Briefly, 1×10⁵ cells were incubated with 10 μg/ml of Controlantibody I, or an isotype control for 30 min on ice in antibody dilutionbuffer. Following two washes with antibody dilution buffer, cells wereincubated with 10 μg/ml of PE conjugated anti-human secondary antibodiesfor 30 min on ice. Following two additional washes, samples were run ona Hypercyt® cytometer and analyzed in ForeCyt™ (IntelliCyt, Albuquerque,N. Mex.). The mean fluorescence intensities (MFI) were expressed as foldchange above isotype control levels (background). FACS binding confirmedthat Control I selectively bound to 293/hPRLR expressing cells with MFIsthat were 30 fold above background (isotype ctrl) levels and less than 2fold binding on parental cells.

Cell surface copy number of PRLR was also quantitatively determined onT47D, MCF7 and MCF7/hPRLR-overexpressing cell lines. Briefly, 1×10⁵cells were incubated with 100 nM of the anti-PRLR antibodyH1H6953N-Alexa647 for 30 min on ice in antibody dilution buffer.Following two washes with antibody dilution buffer, samples were run ona Hypercyt® cytometer (IntelliCyt, Albuquerque, N. Mex.) and the meanfluorescence intensities (MFI) were determined in ForeCyt™ (IntelliCyt,Albuquerque, N. Mex.). The MFI for each cell line was then converted toAlexa647 molecules of equivalent soluble fluorescence (MESF) via theQuantum Alexa Fluor 647 MESF kit according to manufacturer instructions(Bangs Laboratories, Inc, Fishers, Ind.). The average number offluorophores per H1H6953N-A647 protein (F/P ratio) was determined viathe Simply Cellular anti-Human IgG kit according to manufactureinstructions (Bangs Laboratories, Inc, Fishers, Ind.). The MESF valueswere divided by the F/P ratio to determine the PRLR cell surface copynumber or H1H6953N antigen binding capacity on each cell line. Usingthis method, it was determined that the approximate cell surface copynumber of PRLR on the various cell lines was as follows: T47D=27,000;MCF7=3,000; and MCF7/hPRLR=190,000.

Next, the anti-PRLR antibodies of the present invention were tested viaFACS for selective binding to the engineered overexpressing PRLR HEK293cell lines, as well as to non-expressing HEK293 cells and a native PRLRexpressing cell line (T47D). Results are shown in Table 5.

TABLE 5 FACS Cell Surface Binding of Anti-PRLR Antibodies FACS CellBinding (Fold Above Background) HEK293/ HEK293/ HEK293/ Antibody HEK293hPRLR mfPRLR mPRLR T47D unstained 1 1 1 1 1 Secondary 1 1 1 1 1 onlyControl I 1 31 13 7 30 *H1H6762P 1 36 26 2 38 *H1H6765P 2 38 27 1 40H1H6774P 1 1 29 1 39 *H1H6781P 1 37 26 1 40 H1H6782P 3 41 32 4 43*H1H6783P 1 37 27 1 38 *H1H6785P 1 37 26 2 40 *H1H6790P 1 37 24 1 34*H1H6792P 1 37 28 3 37 H1H6793P 2 2 32 2 43 H1H6795P 1 1 22 1 34H1H6797P 1 1 25 2 38 *H1H6800P 1 35 29 1 41 H1H6801P 7 47 88 56 45*H1H6803P 1 39 28 1 39 H1H6804P 1 1 29 1 34 *H1H6807P 2 32 27 2 34*H1H6953N 2 37 29 2 40 *H1H6958N2 1 35 30 1 37 H1H6959N2 1 6 11 2 9*H1H6960N 1 29 23 1 33 *H1H6966N 1 29 18 1 31 *H1H6967N 1 38 28 2 41*H1H6975N 1 38 29 1 46 H1H6976N 1 8 29 1 37 Isotype Ctrl I 1 1 1 1 NAIsotype Ctrl II 1 1 NA NA 1 *Denotes antibodies with specific binding onHEK293/hPRLR, HEK293/mfPRLR and T47D and less than 2-fold binding onHEK293 parental cells.

As shown in Table 5, a majority of the anti-human PRLR antibodiesspecifically bound to HEK293/PRLR cells at >25-fold above backgroundlevels with negligible binding to parental cells. Antibodies that boundto human PRLR were similarly shown to bind to monkey (macacafascicularis) PRLR on HEK293/mfPRLR cells. Antibodies that wereidentified to be strong binders to HEK293/hPRLR cells were similarlyshown to be robust binders to native PRLR expressing T47D cells.Cross-reactivity to rodent PRLR was not observed.

In summary, antibodies of this invention displayed strong binding tohuman and monkey PRLR on engineered cell lines as well as endogenouslyexpressed PRLR.

Example 4B. Anti-PRLR Antibodies are Internalized by PRLR-ExpressingCells In Vitro

In this Example, the internalization of anti-PRLR antibodies byPRLR-expressing cells (T47D) was assessed. Briefly, 20,000 T47D cellswere seeded in collagen coated 96 well plates. The next day, cells wereincubated with anti-human PRLR antibodies (10 μg/ml) for 30 min on icefollowed by two PBS washes. Cells were then incubated with alexa488conjugated anti-hFc Fab secondary antibodies for 30 minutes on icefollowed by two additional PBS washes. Antibodies were allowed tointernalize for 1h at 37° C. in internalization buffer (PBS+2% FBS) orremained at 4° C. Cells were fixed in 4% formaldehyde, nuclei werestained with DRAQ5 (Cell signaling), and images were acquired on theImageXpress micro XL (Molecular Devices). Whole cell alexa488 intensityat 37° C. (Binding) and the alexa488 intensity in the intracellularvesicles at 37° C. (Internalization) were determined via Columbus imageanalysis software (PerkinElmer). The intensities are expressed as apercentage of the strongest internalizing antibody, H1H6975N, and aresummarized in Table 6.

TABLE 6 Cell Line: T47D [mAb] 1 μg/mL (0.67 nM) % Internalization %Total Binding Antibody relative to Control I relative to Control Ianti-PRLR 100.0 100.0 control I H1H6975N 214.2 225.9 H1H6800P 198.3186.8 H1H6803P 190.5 205.5 H1H6762P 186.3 176.7 H1H6765P 186.3 191.3H1H6793P 179.9 177.9 H1H6782P 179.3 209.9 H1H6976N 169.5 180.4 H1H6785P169.1 168.0 H1H6958N2 169.0 161.5 H1H6967N 168.6 158.6 H1H6781P 165.1166.2 H1H6774P 162.3 173.7 H1H6783P 160.7 165.8 H1H6792P 155.9 110.6H1H6953N 153.9 164.2 H1H6795P 150.7 123.4 H1H6801P 148.9 155.7 H1H6807P147.0 152.7 H1H6790P 146.9 122.8 H1H6797P 145.2 151.2 H1H6804P 138.9149.6 H1H6966N 137.2 111.3 H1H6960N 120.0 89.7 H1H6959N2 15.3 2.0

With the exception of H1H6959N2, all tested antibodies bound T47D andnearly 100% of all bound mAbs internalized within 1h. The totalinternalized antibody intensity for most antibodies was greater than theanti-human PRLR control antibody (Control I).

Example 5. Anti-PRLR Antibodies Inhibit PRL-Mediated Receptor Activationin Cells Expressing Human PRLR

The ability of anti-PRLR antibodies to block prolactin (PRL)-mediatedreceptor activation was examined in a luciferase-based reporter assay.The endocrine hormone PRL binds to the extracellular domain of itscognate receptor PRLR, triggering rapid homodimerization and activatingseveral downstream signaling cascades.

In this example, an engineered cell line was used to determine theability of anti-PRLR antibodies to block ligand activation of the PRLRreceptor. Briefly, HEK293/hPRLR/STAT5-Luc cell lines with stableincorporation of a human PRLR expression cassette and theSTAT5-dependent luciferase reporter were generated via sequential roundsof Lipofectamine® 2000-mediated transfection (LifeTechnologies,Carlsbad, Calif.). Cells were selected for at least two weeks in thepresence of 500 pg/mL G418 (hPRLR) and 100 pg/mL hygromycin B(STAT5-Luc). The STAT5-Luc assay utilized 2×10⁵ HEK293/hPRLR/STAT5-Luccells seeded in complete growth medium on PDL-coated 96 well platesgrown overnight at 37° C., 5% CO₂. To generate antibody inhibitioncurves, cells were incubated (6 hr at 37° C.) with serially dilutedanti-human PRLR antibodies (100 nM to 24 pM) in the presence of 5 nMconstant PRL before recording signal. PRL dose response curves weregenerated by the addition of serially diluted PRL (100 nM to 24 pM) tocells and recording signal after a 6 hr (37° C.) incubation in theabsence of antibodies. The ability of the antibodies to activate PRLR inthe absence of ligand was also assessed.

Luciferase activity was measured with ONE-Glo™ reagent (Promega,Madison, Wis.). Relative light units (RLUs) were measured on a Victorluminometer (Perkin Elmer, Shelton, Conn.). EC₅₀/IC₅₀ values weredetermined from a four-parameter logistic equation over an 8-pointresponse curve using GraphPad Prism. Percent blocking and percentactivation are reported for the highest antibody dose. Results are shownin Table 7.

TABLE 7 IC₅₀ and Percent Blocking of PRL-Mediated Signaling by Anti-PRLRAntibodies IC₅₀ of Blocking Percent Blocking Antibody 5 nM PRL [M] at100 nM Antibody H1H6762P 2.19E−11 100 H1H6765P 3.30E−11 100 H1H6774P NB0 H1H6781P 2.70E−11 100 H1H6782P NB 0 H1H6783P ND 59 H1H6785P 3.45E−10100 H1H6790P 2.06E−10 100 H1H6792P 5.70E−10 100 H1H6793P ND 65 H1H6795PNB 0 H1H6797P NB 0 H1H6800P 3.55E−10 100 H1H6801P 1.40E−10 100 H1H6803P6.27E−10 100 H1H6804P 7.89E−09 100 H1H6807P 6.00E−10 100 H1H6953N1.05E−10 100 H1H6958N2 1.98E−10 100 H1H6959N2 ND 52 H1H6960N 1.58E−09100 H1H6966N ND 54 H1H6967N 7.68E−10 100 H1H6975N 2.41E−10 100 H1H6976NND 23 Control I 1.33E−09 100 NB: Not blocking; ND: Not determined due toincomplete blocking

As summarized in Table 7, a majority of the antibodies of this inventioninhibited activation of the STAT5 reporter, with IC₅₀ values rangingfrom 22 pM to 8 nM. All inhibitory antibodies blocked activation tobaseline levels (100 percent blocking). Additionally, the antibodiestested in this assay did not activate STAT5 in the absence of PRLligand.

In summary, the data of this Example show that a majority of theanti-PRLR antibodies of the invention block PRL-mediated receptoractivation. Additionally, a majority of the antibodies inhibit receptoractivation more potently than the anti-PRLR Control I antibody. Forexample, several anti-PRLR antibodies of the present invention blockedprolactin-mediated signaling with IC₅₀ values of less than about 1.3 nM.On the other hand, certain anti-PRLR antibodies of the invention,despite being able to bind PRLR, did not exhibit prolactin blockingactivity. Such non-blocking anti-PRLR antibodies may find uses invarious therapeutic contexts where PRLR targeting is desired withoutinterfering with normal prolactin-mediated signaling.

Example 6. Preparation and Characterization of Anti-PRLR Antibody DrugConjugates

Selected anti-PRLR antibodies were conjugated to the maytansinoid DM1through an SMCC linker using methods similar to those set forth in U.S.Pat. No. 5,208,020 and US Patent Application Publication No.2010/0129314, the disclosures of which are incorporated by referenceherein in their entireties. The conjugates were purified by sizeexclusion chromatography and sterile filtered. All starting materialsand solvents were purchased commercially and used without purification,unless otherwise noted.

Protein and linker/payload concentrations were determined by UV spectralanalysis and MALDI-TOF mass spectrometry. Size-exclusion HPLCestablished that all conjugates used were >95% monomeric, and RP-HPLCestablished that there was <0.5% unconjugated linker payload. Yields arereported in Table 8 and 9 based on protein. All conjugated antibodieswere analyzed by UV for linker payload loading values according toHamblett et al, 2004, Clinical Cancer Research 10(20):7063-7070, and bymass difference, native versus conjugated.

TABLE 8 Protein Concentrations for Anti-PRLR Unconjugated Antibodiesϵ252 nm ϵ280 nm (cm⁻¹ M⁻¹) (cm⁻¹ M⁻¹) Compound SMCC-DM1 26790 5700Antibody (unconjugated) H1H6958N2 74462 195440 H1H6959N2 77485 209420H1H6960N 84926 214460 H1H6953N 80673 220420 H1H6975P 81120 199804Isotype Control 84723 218360

TABLE 9 Antibody Linker/Payload Concentrations for Anti-PRLR-SMCC-DM1Conjugates Antibody Payload:Antibody Payload:Antibody Conjugate MolarRatio (UV) Molar Ratio (MS) Yield % H1H6958N2-DM1 4.0 3.4 64H1H6959N2-DM1 3.8 3.3 64 H1H6960N-DM1 3.6 3.0 64 H1H6953N-DM1 3.2 2.7 52H1H6803P-DM1 ND 3.1 55 H1H6762P-DM1 ND 2.9 70 H1H6765P-DM1 ND 2.3 55H1H6782P-DM1 ND 2.8 65 H1H6793P-DM1 ND 3.8 55 H1H6975P-DM1 3.0 3.4 60H1H6800P-DM1 3.0 3.2 50 Isotype Control-DM1 3.3 3.3 80 ND: notdetermined

This Example illustrates the conjugation of anti-PRLR antibodies of thepresent invention with DM1 through an SMCC linker. The payload: antibodymolar ratio was calculated to be from about 2.3 to about 3.8 for theconjugated antibodies of this Example. Percent yields for the antibodiesof the invention ranged from around 50% to 70%.

Example 7. Anti-PRLR Antibody-Drug Conjugates Effectively Kill Cellswith Low-to-Moderate PRLR Expression Levels as Well as Cells with HighPRLR Expression Levels

To determine the relative cell-killing potency of anti-PRLR ADCs of theinvention compared to a similar anti-ErbB2 ADC, cell-killing assays wererun on multiple cells lines expressing either PRLR, ErbB2 or acombination of both receptors.

PRLR-overexpressing cells, including HEK293, PC3, MCF7 and NCI-N87, weregenerated to assess the ability of anti-PRLR conjugated antibodies toreduce cell viability. For comparative purposes, PC3 and T47D cells withoverexpressed ErbB2 were also generated, as well as an MCF7 cell lineover-expressing both hPRLR and hErbB2. Briefly, Lipofectamine®2000-mediated transfection methodology was utilized to generate HEK293cells expressing human PRLR (HEK293/hPRLR) or human ErbB2(HEK293/hErbB2). Lipofectamine LTX with Plus Reagent was used togenerate PC3 cells expressing human PRLR (PC3/hPRLR) or human ErbB2(PC3/hErbB2). Lentiviral-mediated transduction was utilized to generateMCF7 cells expressing human PRLR (MCF7/hPRLR), NCI-N87 cells expressinghuman PRLR (NCI-N87/hPRLR), T47D cells over-expressing human ErbB2(T47D/hErbB2), and MCF7 cells expressing both human PRLR and human ErbB2(MCF7/hPRLR/hErbB2). All lines were selected for at least two weeks incomplete growth media plus appropriate selection reagents. Stablyexpressing populations were enriched for PRLR expression via FACS usingthe anti PRLR antibody Control I.

Cell surface expression of PRLR and ErbB2 was analyzed via FACS usingeither the Control I anti-PRLR antibody or Control II anti-HER2antibody, respectively. Additionally, endogenous PRLR cell surfaceexpression on the T47D#11 cell line, a variant of the T47D line selectedfor more aggressive in vivo tumor growth, was also determined.Approximately 1×10⁶ cells were incubated with 10 μg/ml of anti-PRLRControl Antibody (Control I), an anti-ErbB2 control antibody (ControlII), or an isotype control for 30 min on ice in antibody dilutionbuffer. Following two washes with antibody dilution buffer, cells wereincubated with 10 μg/ml of PE conjugated anti-human secondary antibodiesfor 30 min on ice. Following two additional washes, samples were run onthe Accuri C6 (BD) cytometer and analyzed with FlowJo software (TreeStar, Inc., Ashland, Oreg.). Relative expression level results are shownin Table 10.

TABLE 10 Human PRLR Cell Surface Expression (Engineered & EndogenousLines) FACS Binding (MFI FOLD ABOVE ISOTYPE CONTROL) Anti- Anti- PRLRErbB2 Secondary Isotype (Con- (Con- Cell Line Unstained alone Ctrl trolI) trol II) 293 1X 1X 1X 1X 28X 293/hErbB2 1X 1X 1X 1X 215X  293/hPRLR1X 1X 1X 18X  18X PC3 1X 1X 1X 1X 41X PC3/hErbB2 1X 1X 1X 1X 238X PC3/hPRLR 1X 1X 1X 13X  31X T47D 1X 1X 1X 12X  87X T47D#11 1X 1X 1X 10X ND T47D/hErbB2 1X 1X 1X 12X  437X  SK-BR-3 1X 1X 1X 1X 600X  MCF7 1X 1X1X 3X 42X MCF7/hPRLR 1X 1X 1X 55X  36X MCF7/hPRLR/ 1X 1X 1X 55X  349X hErbB2 NCI-N87 1X 1X 1X 1X 1,400X   NCI-N87/hPRLR 1X 1X 1X 6X 1,400X  

In general, exogenous PRLR surface expression ranged from 6-fold to55-fold over background, with most engineered cells exhibiting 12-foldto 18-fold PRLR expression over background. Endogenous PRLR expressionwas 3-fold over background in MCF7 cells but was not detected inparental HEK293, PC3 and NCI-N87 lines. Endogenous PRLR expression was12-fold over background in the T47D cell line and 10-fold overbackground in the T47D#11 variant cell line. ErbB2 expression wasdetected in all PRLR-expressing cell lines, and ranged from 18-fold to1400-fold above background.

Next, the ability of anti-PRLR-DM1 antibody-drug conjugates (i.e.,anti-PRLR antibodies conjugated to DM1 via a non-cleavable linker[SMCC]) to reduce cell viability was determined using in vitro cellbased assays. Cells were seeded in PDL-coated 96 well plates at 1500 to10000 cells per well in complete growth media and allowed to growovernight. For cell viability curves, ADCs or free DM1 (as the methyldisulfide derivative DM1-SMe) were added to the cells at finalconcentrations ranging from 500 nM to 5 pM and incubated for 3 days. The293, PC3 and T47D cells were incubated with CCK8 (Dojindo, Rockville,Md.) for the final 1-3 hours and the absorbance at 450 nm (OD₄₅₀) wasdetermined on a Flexstation3 (Molecular Devices, Sunnyvale, Calif.).MCF7 cells were treated with Hoechst 33342 nuclear stain while beingfixed with 4% formaldehyde. Images were acquired on the ImageXpressmicro XL (Molecular Devices, Sunnyvale, Calif.) and nuclear counts weredetermined via Columbus image analysis software (Perkin Elmer, Shelton,Conn.). Background OD₄₅₀ values (PC3, 293, and T47D) or nuclear counts(MCF7) from digitonin (40 nM) treated cells was subtracted from allwells and viability was expressed as a percentage of the untreatedcontrols. IC₅₀ values were determined from a four-parameter logisticequation over a 10-point response curve (GraphPad Prism). IC₅₀ valuesand percent cell killing are shown in Tables 11 and 12.

TABLE 11 Cell Kill Potency of Anti-PRLR-DM1 Antibody-Drug ConjugatesAntibody-Drug 293 293/PRLR PC3 PC3/PRLR Conjugate IC₅₀ % Kill IC₅₀ %Kill IC₅₀ % Kill IC₅₀ % Kill DM1 (free drug) 0.27 98 0.36 97 0.47 910.59 80 H1H6953N-DM1 100 88 0.28 95 110 78 0.86 67 H1H6958N2-DM1 75 980.10 95 150 83 0.43 83 H1H6959N2-DM1 75 100 4.82 95 150 79 11.6 82H1H6960N-DM1 75 100 0.38 95 150 80 1.13 82 H1H6975N-DM1 100 87 ND ND 20071 2.70 68 H1H6762P-DM1 100 89 ND ND 250 78 0.98 66 H1H6765P-DM1 100 86ND ND 200 70 0.57 70 H1H6782P-DM1 100 86 ND ND 300 67 0.92 64H1H6793P-DM1 100 84 ND ND 200 72 3.65 65 H1H6800P-DM1 150 85 ND ND 15072 1.37 68 H1H6803P-DM1 300 7 ND ND 150 72 2.32 70 Control I-DM1 100 920.28 95 >100 79 7.28 80 Isotype ctrl-DM1 100 60 100 93 125 78 110 63IC₅₀ values are in nM; ND: not determined

TABLE 12 Cell Kill Potency of Anti-PRLR-DM1 Antibody-Drug Conjugates(continued) T47D MCF7 MCF7/PRLR Antibody-Drug % % % Conjugate IC₅₀ KillIC₅₀ Kill IC₅₀ Kill DM1 (free drug) 0.45 86 1.17 83 1.47 88 H1H6953N-DM11.64 79 100 55 0.29 76 H1H6958N2-DM1 1.34 71 100 77 0.19 77H1H6959N2-DM1 48.90 71 100 82 0.47 80 H1H6960N-DM1 5.10 71 100 77 0.2978 H1H6975N-DM1 6.90 80 150 73 0.49 87 H1H6762P-DM1 12.60 80 110 64 0.4683 H1H6765P-DM1 1.63 78 150 68 0.18 84 H1H6782P-DM1 4.19 74 120 57 0.6585 H1H6793P-DM1 11.10 53 125 67 0.54 87 H1H6800P-DM1 3.20 79 150 65 0.4085 H1H6803P-DM1 8.61 77 150 60 0.44 84 Control I-DM1 24.50 65 100 460.59 75 Isotype ctrl-DM1 150 61 120 66 100 84 IC₅₀ values are in nM

As summarized in Tables 11 and 12, several anti-PRLR-DM1 antibody-drugconjugates potently reduced cell viability in multiple cell backgrounds,with IC₅₀ values as low as 100 pM. An exemplary anti-PRLR conjugatedantibody, H1H6958N2-DM1, reduced the cell viability of HEK293, PC3 andMCF7-PRLR expressing cells with sub-nM IC₅₀s ranging from 100 pM to 460pM, and killed endogenously expressing T47D cells with an IC₅₀ of 1.3nM. The similarly conjugated anti-PRLR Control I antibody (ControlI-DM1) was several fold less potent than H1H6958N2-DM1 across all celllines. Non-binding isotype controls and unconjugated antibodies had noimpact on cell viability.

Additionally, the impact of the PRLR ligand, PRL, on PRLR-SMCC-DM1 cellkill in T47D cells was assessed. T47D cells were incubatedsimultaneously with PRL (15 nM) and either a non-blocking anti PRLRantibody (H1H6782P) or a receptor blocking antibody (H1H6958N2). Resultsare summarized in Table 13.

TABLE 13 Cell Kill Potency of Anti-PRLR-DM1 Antibody-Drug Conjugates inthe Presence of PRLR Ligand (PRL) HEK293 T47D IC₅₀ % IC₅₀ % Treatment(nM) Kill (nM) Kill Me-SS-May 0.6 94 0.30 100 Free DM1 IsotypeControl-SMCC-DM1 100 78 300 94 Isotype Control-SMCC-DM1 + 170 82 97 8915 nM PRL H1H6958N2-SMCC-DM1 90 88 1.0 100 H1H6958N2-SMCC-DM1 + 110 863.0 100 15 nM PRL H1H6782P-SMCC-DM1 90 83 1.0 98 H1H6782P-SMCC-DM1 + 7078 2.0 97 15 nM PRL

As shown in Table 13, the presence of PRL had only a modest impact onPRLR ADC-mediated cell kill with an observed 2-3 fold reduction in thecell kill potency of the tested mAbs.

The potency of anti-PRLR conjugated antibodies compared with a similarlyconjugated antibody to the co-expressed ErbB2 cell surface target wasalso assessed. Both PRLR and ErbB2 are expressed in a majority of breastcancers, and anti-ErbB2 antibodies conjugated to DM1 have shown clinicalefficacy in targeting ErbB2 (+) breast cancer (Hurvitz et al; 2013). AnErbB2 Control Antibody (Control II) conjugated to DM1 (Control II-DM1)was tested in in vitro viability assays in the cell lines generatedabove. Cell kill potency of conjugated anti-PRLR antibodies compared tothe anti-ErbB2 conjugated antibody is summarized in Tables 14-17.

TABLE 14 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates Antibody-Drug 293 293/ErbB2 293/PRLR ConjugateIC₅₀ % Kill IC₅₀ % Kill IC₅₀ % Kill DM1 0.5 95 0.26 100 0.82 95 (freedrug) H1H6953N-DM1 100 92 120 98 0.43 93 (Anti PRLR-DM1) Control II-DM1100 89 2.0 98 100 88 (Anti ErbB2-DM1) Isotype Control-DM1 150 86 110 98150 86 PRLR expression  1x  1X 18X ErbB2 expression 28X 215X 18X IC₅₀values are in nM

TABLE 15 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates (continued) Antibody-Drug PC3 PC3/ErbB2PC3/PRLR Conjugate IC₅₀ % Kill IC₅₀ % Kill IC₅₀ % Kill DM1 0.44 88 0.4285 0.24 82 (free drug) H1H6953N-DM1 100 80 80 73 0.68 76 (Anti PRLR-DM1)Control II-DM1 90 80 1.1 80 100 62 (Anti ErbB2-DM1) Isotype Control-DM185 82 90 71 100 62 PRLR expression  1X  1X 13X ErbB2 expression 41X 238X31X IC₅₀ values are in nM

TABLE 16 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates (continued) Antibody-Drug T47D T47D/ErbB2SK-BR-3 MCF7 Conjugate IC₅₀ % Kill IC₅₀ % Kill IC₅₀ % Kill IC₅₀ % KillDM1 0.21 80 0.18 71 0.39 74 0.54 78 (free drug) H1H6953N-DM1 1.3 78 2.480 100 77 82 74 (Anti PRLR-DM1) Control II-DM1 100 59 1.18 81 0.48 81 8068 (Anti ErbB2-DM1) Isotype Control- 100 62 120 75 110 76 100 65 DM1PRLR expression 12X  12X  1X  3X ErbB2 expression 87X 437X 600X 42X IC₅₀values are in nM

TABLE 17 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates (continued) MCF7/ MCF/PRLR + NCI-N87/Antibody-Drug PRLR ErbB2 NCI-N87 PRLR Conjugate IC₅₀ % Kill IC₅₀ % KillIC₅₀ % Kill IC₅₀ % Kill DM1 1.85 79 0.82 77 0.84 95 1.45 98 (free drug)H1H6953N-DM1 0.33 76 0.33 77 90 83 2.9 94 (Anti PRLR-DM1) Control II-DM1100 56 0.63 76 0.22 95 0.66 94 (Anti ErbB2-DM1) Isotype Control-DM1 15061 100 70 90 85 85 88 PRLR expression 55X  55X   1X   6X ErbB2expression 36X 349X 1400X 1400X IC₅₀ values are in nM

Anti-PRLR-DM1 antibodies effectively killed cells even with relativelylow levels of PRLR expression. For example, H1H6953N-DM1 (anti-PRLR-DM1)inhibited the growth of T47D cells (expressing PRLR at only 12× abovebackground) with an IC₅₀ of 1.3 nM and showed 78% killing. This sameantibody also inhibited the growth of 293/hPRLR cells (expressing PRLRat 18× above background) with and IC₅₀ of 0.43 nM and showed 93%killing. Equivalent killing with the anti-ErbB2-DM1 antibody (“controlII”) was observed only in cells that express the target antigen atlevels greater than about 200× to about 400× above background (see e.g.,PC3/hErbB2, expressing ErbB2 at 238× above background and T47D/hErbB2,expressing ErbB2 at 437× above background). Therefore, these datasuggest that anti-PRLR antibody-drug conjugates can effectively targetand kill tumor cells with relatively low levels of PRLR expression,while anti-ErbB2 antibody drug conjugates are effective only againsttumors with very high ErbB2 expression levels.

Finally, the potency of anti-PRLR antibodies conjugated to DM1 via thenon-cleavable linker SMCC was compared to the cell killing potency ofanti-PRLR antibodies conjugated to MMAE via the cleavable linker:mc-vc-PAB (available from Concortis, San Diego, Calif.). Cells used inthis experiment were PC3, PC3/hPRLR, MCF7/ATCC and MCF7/PRLR. Resultsare shown in Table 18.

TABLE 18 Anti-PRLR ADC Cell Kill Potency PC3/ MCF7/ MCF7/ PC3 hPRLR ATCCPRLR IC₅₀ % IC₅₀ % IC₅₀ % IC₅₀ % (nM) Kill (nM) Kill (nM) Kill (nM) KillFree DM1 0.5 90 1.0 70 3.0 80 1.9 86 Free MMAE 1.4 90 1.0 77 2.4 83 1.595 Isotype 95 79 100 79 100 71 100 76 Control I-SMCC- DM1 Isotype 200 33145 55 143 23 143 21 Control I-mc-VC- PAB-MMAE H1H6953N- 90 81 0.4 83 8070 0.1 80 SMCC-DM1 H1H6953N- 130 49 0.2 80 150 42 0.1 85 mc-VC-PAB- MMAEH1H6958N2- 110 81 0.5 79 90 72 0.2 83 SMCC-DM1 H1H6958N2- 100 39 0.5 82150 53 0.2 88 mc-VC-PAB- MMAE H1H6765P- 80 80 0.3 83 90 71 0.1 81SMCC-DM1 H1H6765P- 150 29 0.40 85 150 38 0.2 86 mc-VC-PAB- MMAE

As shown in Table 18, nearly equivalent cell killing in PC3/hPRLR andMCF7/hPRLR cell lines was observed for both the non-cleavable DM1 ADCs(H1H6953-SMCC-DM1, H1H6958N2-SMCC-DM1, and H1H6765-SMCC-DM1) and for thecleavable MMAE ADCs (H1H6953-mc-vc-PAB-MMAE, H1H6958N2-mc-VC-PAB-MMAE,and H1H6765-mc-VC-PAB-MMAE).

Additional toxins (DM4, MeNHC3-May, and MMD) conjugated to anti-PRLRantibodies were also tested in T47D and MCF7/hPRLR cell lines, andresults are summarized in Tables 19 (293 and T47D cell killing) and 20(MCF7/ATCC and MCF7/PRLR cell killing). (ND=not detected).

TABLE 19 Anti-PRLR Antibody Drug Conjugates-Cell Killing Properties (293and T47D Cell Lines) Cell Line 293 T47D IC₅₀ % IC₅₀ % Antibody LinkerDrug nM Kill nM Kill Free DM1 1.2 95 1.5 100 (Me-SS- May) MMAE 0.9 100 1100 DM4 0.6 100 0.5 100 MMD 0.9 100 2 100 MeNHC3- 60 90 90 100 MayIsotype SMCC DM1 150 80 140 50 Control I mc-VC-PAB MMAE 300 30 300 20MMD 300 70 140 70 MeNHC3- 300 30 300 30 May SPDB DM4 50 90 30 100H1H6953N SMCC DM1 100 90 1.5 100 mc-VC-PAB MMAE ND ND 1.0 80 MMD 110 701.0 90 MeNHC3- 300 20 1.0 90 May H1H6958N2 SMCC DM1 100 90 2 100mc-VC-PAB MMAE ND ND 1 90 SPDB DM4 ND ND 1 100 H1H6975P SMCC DM1 80 90 2100 mc-VC-PAB MMD 110 80 3 90 H1H6782P SMCC DM1 120 80 3 100 mc-VC-PABMMD 230 70 2 90 mc-VC-PAB MeNHC3- 60 90 2 90 May H1H6765P SMCC DM1 85100 1 90 mc-VC-PAB MMAE ND ND 1 90

TABLE 20 Anti-PRLR Antibody Drug Conjugates-Cell Killing Properties(MCF7/ATCC and MCF7/PRLR Cell Lines) Cell Line MCF7/ MCF7/ ATCC PRLRIC₅₀ % IC₅₀ % Antibody Linker Drug nM Kill nM Kill Free DM1 1.3 80 0.880 Me-SS-May MMAE 2.4 80 1.5 95 DM4 1.3 90 2 90 MMD 2 90 0.4 90MeNHC3-May 100 70 50 80 Isotype SMCC DM1 300 60 150 70 Control Imc-VC-PAB MMAE 140 20 140 20 MMD 300 40 70 75 MeNHC3-May 300 30 200 55SPDB DM4 70 80 80 90 H1H6953N SMCC DM1 90 70 0.3 80 mc-VC-PAB MMAE 15040 0.1 85 MMD 300 50 0.2 90 MeNHC3-May 150 60 0.2 80 H1H6958N2 SMCC DM170 70 0.2 80 mc-VC-PAB MMAE 150 50 0.2 90 SPDB DM4 25 80 0.8 90 H1H6975PSMCC DM1 200 70 0.2 80 mc-VC-PAB MMD 150 50 0.2 90 H1H6782P SMCC DM1 25070 0.3 80 mc-VC-PAB MMD 200 50 0.3 90 mc-VC-PAB MeNHC3-May 250 50 0.2 80H1H6765P SMCC DM1 150 70 0.3 90 mc-VC-PAB MMAE 150 40 0.2 90

All tested anti PRLR ADCs, regardless of the toxin and linker utilized,specifically killed the tested cells. T47D cell viability IC₅₀s rangedfrom 0.6 nM to 3.4 nM and MCF7/hPRLR cell viability IC50s ranged from0.2 nM to 0.8 nM.

Example 8. Anti-PRLR Antibody-Drug Conjugates Effectively Inhibit TumorGrowth In Vivo

To determine the in vivo efficacy of the anti-PRLR-DM1 antibody-drugconjugates, studies were performed in immunocompromised mice bearingPRLR+ breast cancer xenografts.

Briefly, 20×10⁶ MCF7/PRLR cells (ATCC HTB-22 transfected with fulllength hPRLR as previously described) were implanted subcutaneously intothe left mammary fat pad of female NCr nude mice. In other studies,10×10⁶ PC3/PRLR (ATCC CRL-1435 transfected with full length hPRLR aspreviously described) were implanted subcutaneously into the left flankof male SCID mice. Additionally, 10×10⁶ parental T47D (ATCC HTB-133) or7.5×10¹⁰ T47D#11 cells (ATCC HTB-133 serially passaged in vivo asdescribed below) were subcutaneously implanted into the left flank offemale CB17 SCID mice. All mice were obtained from Taconic (Hudson,N.Y.). Each bolus of cells was supplemented with a 90-day estrogenrelease pellet (1.7 mg/pellet; Innovative Research America, SarasotaFla.). Once tumors had reached an average volume of 250 mm³, mice wererandomized into groups of seven and dosed with anti-PRLR antibody-drugconjugates of the invention or control reagents. Control reagentsincluded PBS vehicle, free methyl-disulfide DM1 (DM1-SMe) and isotypeControl 1-DM1.

In multi-dose studies, mice were dosed once a week for a total of threeweeks with tumor volumes and body weights being monitored twice weeklythroughout the study. Test ADCs were dosed at 5 and/or 15 mg/kg in themulti-dose studies. In single-dose studies, mice received a single doseof test ADC, and tumor volumes and body weights were monitored twiceweekly throughout the study. Test ADCs were dosed at 1, 2.5, 5, and 15mg/kg in the single-dose studies. Average tumor size as well as tumorgrowth inhibition relative to the vehicle treated group were calculatedfor each group. Tumors were measured with calipers twice a week untilthe average size of the vehicle group reached 1000 mm³. Tumor size wascalculated using the formula (length×width²)/2. Tumor growth inhibitionwas calculated according to the following formula:(1−((T_(final)−T_(initial))/(C_(final)−C_(initial))))*100, where T(treated group) and C (control group) represent the mean tumor mass onthe day the vehicle group reached 1000 mm³. Animals were observed to Day52. Results are summarized in Tables 21-25 (multi-dose) and Table 26(single dose). (NT=not tested in the particular experiment shown).

TABLE 21 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -MCF7/PRLR tumors (TRIAL #1) [NCr Nude mice - data collected at Day 52]Final Tumor Average Dose size mm³ Tumor Growth Treatment Group (mg/kg)(mean ± SEM) Inhibition (%) Vehicle — 1068 ± 384  — Free DM1 0.2 625 ±141 57 Isotype control Ab-DM1 5 NT NT 15 300 ± 141 96 H1H6958N2 15 483 ±46  74 H1H6958N2-DM1 5 51 ± 33 128 15 0 ± 0 133 H1H6953N 15 421 ± 23  79H1H6953N-DM1 5 107 ± 45  120 15 0 ± 0 133 H1H6975N 15 659 ± 144 51H1H6975N-DM1 5 125 ± 46  118 15 0 ± 0 135 H1H6782P 15 NT NT H1H6782P-DM15 15 H1H6765P 15 NT NT H1H6765P-DM1 5 15

TABLE 22 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -MCF7/PRLR tumors (TRIAL #2) [NCr Nude mice - data collected at Day 63]Final Tumor Average Dose size mm³ Tumor Growth Treatment Group (mg/kg)(mean ± SEM) Inhibition (%) Vehicle — 870 ± 211 — Free DM1 0.2 1080 ±451  −33 Isotype control Ab-DM1 5 1106 ± 371  −39 15 712 ± 214 24H1H6958N2 15 766 ± 128 17 H1H6958N2-DM1 5 117 ± 10  116 15 0 ± 0 137H1H6953N 15 NT NT H1H6953N-DM1 5 15 H1H6975N 15 NT NT H1H6975N-DM1 5 15H1H6782P 15 300 ± 83  88 H1H6782P-DM1 5 74 ± 34 123 15 0 ± 0 137H1H6765P 15 737 ± 182 19 H1H6765P-DM1 5 90 ± 56 122 15 0 ± 0 136

TABLE 23 Tuimbr Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -PC3/PRLR tumors (TRIAL #1) [SCID mice - data collected at Day 63] FinalTumor Average Dose size mm³ Tumor Growth Treatment Group (mg/kg) (mean ±SEM) Inhibition (%) Vehicle — 1311 ± 257 — Free DM1 0.2 1361 ± 120 −5Isotype control Ab-DM1 5 1379 ± 128 −7 15 1091 ± 93  19 H1H6958N2 151507 ± 106 −19 H1H6958N2-DM1 5 1247 ± 171 5 15 808 ± 83 46 H1H6953N 151306 ± 127 0 H1H6953N-DM1 5 1058 ± 138 23 15 892 ± 53 39 H1H6975N 151185 ± 97  12 H1H6975N-DM1 5  973 ± 169 31 15 895 ± 63 38 H1H6782P 15 NTNT H1H6782P-DM1 5 15 H1H6765P 15 NT NT H1H6765P-DM1 5 15

TABLE 24 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -PC3/PRLR tumors (TRIAL #2) [SCID mice - data collected at Day 55] FinalTumor Average Dose size mm³ Tumor Growth Treatment Group (mg/kg) (mean ±SEM) Inhibition (%) Vehicle — 1222 ± 99  0 Free DM1 0.2 1147 ± 59  7Isotype control Ab-DM1 5 1052 ± 101 16 15 1049 ± 127 16 H1H6958N2 15 917 ± 253 28 H1H6958N2-DM1 5 566 ± 63 61 15 230 ± 22 94 H1H6953N 15 NTNT H1H6953N-DM1 5 15 H1H6975N 15 NT NT H1H6975N-DM1 5 15 H1H6782P 151154 ± 212 6 H1H6782P-DM1 5 490 ± 63 69 15 321 ± 33 85 H1H6765P 15 1208± 72  1 H1H6765P-DM1 5 489 ± 70 70 15 181 ± 42 98

TABLE 25 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -T47D#11 tumors [SCID mice - data collected at Day 66] Final TumorAverage Dose size mm³ Tumor Growth Treatment Group (mg/kg) (mean ± SEM)Inhibition (%) Vehicle — 1234 ± 88  0 Free DM1 0.2 1433 ± 23  −19Isotype control Ab-DM1 5 1340 ± 176 −9 15 1678 ± 67  −42 H1H6958N2 151259 ± 122 −3 H1H6958N2-DM1 5 168 ± 19 102 15 44 ± 5 112 H1H6953N 15 NTNT H1H6953N-DM1 5 15 H1H6975N 15 NT NT H1H6975N-DM1 5 15 H1H6782P 151537 ± 111 −29 H1H6782P-DM1 5 293 ± 20 90 15 124 ± 36 106 H1H6765P 151278 ± 164 −3 H1H6765P-DM1 5 183 ± 28 100 15  69 ± 12 111

TABLE 26 Tumor Size and Tumor Growth Inhibition Following Single DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -MCF7/PRLR tumors [data collected at Day 55] Final Tumor Average Dosesize mm³ Tumor Growth Treatment Group (mg/kg) (mean ± SEM) Inhibition(%) Vehicle — 710 ± 249 0 Isotype Control Ab-DM1 15 514 ± 86  38H1H6958N2 15 703 ± 160 1 H1H6958N2-1 1 274 ± 142 86 H1H6958N2-1 2.5 172± 53  109 H1H6958N2-1 5 107 ± 26  120 H1H6958N2-1 15 33 ± 23 136

Discussion

In this example, five exemplary anti-PRLR antibodies conjugated to DM1were initially assessed for the ability to reduce MCF7/PRLR and PC3/PRLRtumor volume in multi-dose studies. In the first multi-dose trial (Table21), H1H6958N2-DM1, H1H6953N-DM1 and H1H6975N-DM1 antibodies potentlyinhibited MCF7/PRLR tumor growth at both 5 and 15 mg/kg doses. At thehighest dose, all three DM1 conjugated antibodies reduced tumors toundetectable levels, with a percent reduction in tumor volume of about133-135%. This finding was replicated in a second multi-dose trial(Table 22) when H1H6958N2-DM1 was tested alongside two additionalexemplary anti-PRLR antibodies conjugated to DM1: H1H6782P and H1H6765P.In this second trial, tumor growth was also reduced to undetectablelevels at the highest dose of 15 mg/kg, with percent reduction in tumorvolume of 136-137%. Although treatment with unconjugated anti-PRLRantibodies resulted in moderate reduction of tumor volume (17-79%)compared to the vehicle group, the greatest inhibition in tumor size wasobserved in cohorts treated with antibody-drug conjugates.

Next, the anti-tumor efficacy of these same exemplary anti-PRLR-DM1antibodies was assessed in multi-dose studies in mice bearing PRLRpositive PC3/PRLR xenografts. (Tables 23 and 24). Mice were treatedafter tumors had grown for 21 days. H1H6958N2-DM1, H1H6953N-DM1 andH1H6975N-DM1 all demonstrated inhibition of tumor growth, especially atthe highest dose of 15 mg/kg. (Table 23). Anti-tumor effect wassimilarly observed in a second trial, when H1H6958N2-DM1 was testedalongside H1H6765P-DM1 and H1H6782P-DM1 after 15 days of tumor growth.At the highest dose administered, tumor inhibition across trials rangedfrom 38-98%. (Table 24). In comparison, an Isotype-control conjugated toDM1 produced only 16% tumor inhibition with final tumor volumes notsignificantly different to vehicle controls.

A further assessment of the anti-PRLR ADCs repeatedly dosed at 5 and 15mg/kg was performed in mice bearing T47D#11 xenografts endogenouslyexpressing PRLR. (Table 25). As in other tumor models, dosing wasinitiated when tumor size averaged 200 mm³. Results obtained in thistumor model were consistent with earlier results and clearlydemonstrated the anti-tumor activity of the anti-PRLR antibodiesconjugated to DM1. For example, H1H6958N2-DM1, H1H6765P-DM1 andH1H6782P-DM1 ADCs potently inhibited tumor growth at both the 5 and 15mg/kg dose. At 5 mg/kg anti-PRLR-DM1 conjugated antibodies exhibited89-100% tumor inhibition whereas at 15 mg/kg DM1-conjugated antibodiesresulted in 106-112% tumor growth inhibition. Importantly, unconjugatedanti-PRLR antibodies were not observed to have any anti-tumor efficacyin this endogenous tumor model, indicating the role of the DM1 conjugatein producing anti-tumor efficacy. Again, efficacy of anti-PRLR ADC wasvery specific as control ADC failed to have any effect on tumor growth.

In a final example, anti-PRLR DM1-conjugated antibody H1H6958N2 wasassessed in MCF7/PRLR xenografted mice in a single-dose study. (Table26). As in multiple-dose studies, established tumors were allowed togrow to approximately 200 mm³ before a single dose was administered.Here, H1H6958N2-DM1 was given at 1, 2.5, 5 and 15 mg/kg. As summarizedin Table 26, a dose dependent anti-tumor effect was seen across the widerange used in this study. Anti-tumor effect was observed at all doses,with 1 mg/kg causing a significant decrease in tumor volume relative tovehicle control tumors. Further, although 15 mg/kg of Isotype ControlI-DM1 had some anti-tumor effect (˜38% tumor growth inhibition), dosesof anti-PRLR-DM1 at 2.5 mg/kg and higher significantly reduced tumorvolume (>100% tumor growth inhibition at all doses above 1 mg/kgtested). Single doses of 5 and 15 mg/kg demonstrated anti-tumor efficacycomparable to that observed following repeat dosing at the same level,illustrating the potency of the anti-PRLR ADCs.

In summary, this example illustrates that conjugated anti-PRLRantibodies of the invention are potent inhibitors of tumor growth andare able to reduce tumor size to undetectable levels in the varioustumor models tested.

Example 9. Antibody-Drug Conjugates Against Class-I Cytokine ReceptorsEffectively Kill Cell Lines Expressing Low Levels of Target Antigen

As discussed elsewhere herein, antibody-drug conjugates against PRLReffectively kill PRLR-expressing cell lines, even those that expressrelatively low levels of target antigen. As previously noted, PRLRbelongs to the class I cytokine receptor family, which includes IL-4Rand IL-6R. Similar to PRLR, IL-4R and IL-6R are single-passtransmembrane receptors; IL-4R mediates IL-4 and IL-13 signaling, whileIL-6R mediates IL-6 signaling via a co-complex with the gp130 receptor.In further support of the general concept that ADCs directed againstclass I cytokine receptors may be used to effectively kill cells,including cells that express low-levels of target antigen, thecell-killing ability of ADCs directed against IL-4R and IL-6R wasevaluated.

Cell surface antigen levels on cells that endogenously or recombinantlyexpress IL-4R or IL-6R were first established using FACS. Briefly,approximately 1 million KG-1 (IL-4R⁺), HEK293/IL-4R and Ramos (IL-6R⁺)cells were incubated with exemplary anti-IL-4R (H4H083P2, see U.S. Pat.No. 7,608,693) and anti-IL-6R (VV6A9-5, see U.S. Pat. No. 7,582,298)antibodies for 30 min on ice. After washing, a PE-conjugated anti-humansecondary antibody (10 μg/ml) was added for 30 min followed by a secondwashing step and subsequent analysis on an Accuri C6 cytometer usingFlowJo software (Tree Star, Inc., Ashland, Oreg.). Relative IL-4R andIL-6R cell surface expression levels were calculated as the meanfluorescence intensity (MFI) above isotype control levels. Expressionlevels are summarized in Table 27.

TABLE 27 Relative IL-4R and IL-6R Cell Surface Expression on IL-4R andIL-6R Endogenously or Recombinantly Expressing Cell Lines Receptor CellLine Expression Level; Fold Over Background Expression HEK293HEK293/IL4R KG-1 Ramos IL-4R 2 50 1 4 IL-6R 1 1 7 1

As shown in Table 27, HEK293 and Ramos cells endogenously expressedIL-4R at levels 2-fold and 4-fold over background, respectively, whilethe engineered HEK293/IL-4R cell line expressed IL-4R at levels 50-foldabove background. IL-4R expression was undetectable over background onKG-1 cells. IL-6R expression was detected at 7-fold above backgroundlevels in KG-1 cells, and not on HEK293 or Ramos cell lines.

Next, exemplary anti-IL-4R (H4H083P2) and anti-IL-6R (VV6A9-5)antibodies were conjugated to the cytotoxic drug DM1 and their potencyin cytotoxicity assays was evaluated. Briefly, HEK293/IL-4R, Ramos orKG-1 cell lines, as well as HEK293 parental cells were seeded inPDL-coated 96-well plates at 1500 to 10,000 cells per well. ADCs or freeDM1 (as the methyl disulfide derivative DM1-SMe) were added to the cellsat final concentrations ranging from 300 nM to 15 pM and incubated for 3days. Cells were incubated with CCK8 (Dojindo, Rockville, Md.) for thefinal 1-3 hours and the absorbance at 450 nm (OD₄₅₀) was determined on aFlexstation3 (Molecular Devices, Sunnyvale, Calif.). Background OD₄₅₀values from digitonin (40 nM) treated cells were subtracted from allwells and viability was expressed as a percentage of the untreatedcontrols. IC₅₀ values were determined from a four-parameter logisticequation over a 10-point response curve (GraphPad Prism). Results arepresented in Table 28.

TABLE 28 Cell killing Properties of Anti-IL4R and Anti-IL6R AntibodyDrug Conjugates on IL4R and IL-6R-Expressing Cell Lines HEK293/ HEK293hIL4R KG-1 Ramos Antibody-Drug IC₅₀ % IC₅₀ % IC₅₀ % IC₅₀ % Conjugate(nM) Kill (nM) Kill (nM) Kill (nM) Kill DM1 (Free Drug) 0.27 100 0.33 981.26 89 0.34 100 Isotype control-DM1 70 89 110 91 100 74 60 91Anti-IL-4R-DM1 80 91 0.22 95 90 83 18 91 Anti-IL-6R-DM1 100 88 70 92 3877 70 94 Receptor Expression Levels: Fold over Isotype Ctrl IL-4R 2 50 14 IL-6R 1 1 7 1

As shown in Table 28, anti-IL-4R-DM1 antibody-drug conjugates reducedRamos cell viability with an IC₅₀ value of 18 nM despite an IL-4Rsurface expression of only 4-fold above background levels. IL-4R-DM1ADCs reduced high IL-4R-expressing HEK293/IL-4R viability with an IC₅₀of 0.22 nM. Anti-IL-6R-DM1 antibody-drug conjugates had a modest butreproducible impact on the viability of KG-1 cells (expressing IL-6R ata level of 7-fold above background) with an IC₅₀ value of 38 nM comparedto an IC₅₀ value of 100 nM by an equivalently conjugated isotype controlADC.

In summary, this Example demonstrates that anti-IL-4R and anti-IL-6Rantibody drug conjugates exhibited potent and reproducible cytotoxicityeven on cell lines expressing modest receptor levels. This result issimilar to what was observed with anti-PRLR ADCs where potent cellkilling was obtained even on cells expressing low levels of PRLR (see,e.g., Example 7 herein). Thus, this Example provides further support forthe inventive concept that anti-class I cytokine receptor ADCs ingeneral may be effective therapeutic agents against cell lines andtumors that express class I cytokine receptors even at low levels.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1-28. (canceled)
 29. An isolated antibody or antigen-binding fragment thereof that binds to cell surface-expressed human prolactin receptor (PRLR) and blocks prolactin-mediated signaling in cells expressing human PRLR, comprising (i) a heavy chain variable domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 290 and (ii) a light chain variable domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO:
 298. 30. The antibody or antigen-binding fragment thereof of claim 29, wherein the heavy chain variable domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:
 290. 31. The antibody or antigen-binding fragment thereof of claim 29, wherein the heavy chain variable domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:
 290. 32. The antibody or antigen-binding fragment thereof of claim 29, wherein the light chain variable domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:
 298. 33. The antibody or antigen-binding fragment thereof of claim 29, wherein the light chain variable domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:
 298. 34. An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof of claim 29 conjugated to a cytotoxic agent, wherein the ADC inhibits the growth of cells expressing PRLR.
 35. The ADC of claim 34, wherein the cytotoxic agent is an auristatin, a calicheamicin, a doxorubicin, a duocarmycin, a maytansinoid, or a tubulysin.
 36. The ADC of claim 35, wherein the cytotoxic agent is a maytansinoid.
 37. The ADC of claim 36, wherein the maytansinoid is DM1, DM4, or a derivative thereof.
 38. The ADC of claim 36, wherein the maytansinoid is conjugated to the antibody via a cleavable linker.
 39. The ADC of claim 36, wherein the maytansinoid is conjugated to the antibody via a non-cleavable linker.
 40. The ADC of claim 36, wherein the maytansinoid is conjugated to the antibody via a linker comprising MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), dipeptide site in protease-cleavable linker, ala-phe (alanine-phenylalanine), dipeptide site in protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-Succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), or SIAB (N-Succinimidyl (4-iodo-acetyl)aminobenzoate).
 41. The ADC of claim 37, wherein the maytansinoid is conjugated to the antibody or antigen-binding fragment thereof via an N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.
 42. The ADC of claim 35, wherein the cytotoxic agent is DM1.
 43. The ADC of claim 35, wherein the ADC is a compound of formula I:

wherein Ab is the antibody or antigen-binding fragment thereof.
 44. A pharmaceutical composition comprising the ADC of claim 34 and a pharmaceutically acceptable carrier or diluent. 